MicroRNA-Based Methods and Assays for Osteosarcoma

ABSTRACT

Provided are methods and compositions useful in the diagnosis, treatment, and monitoring of osteosarcoma. Antisense to certain microRNA (miRNA) found to be associated with cancer stem cells (CSCs) or tumor-initiating cells (TICs) of osteosarcoma are useful to suppress tumor growth and metastasis, and prolong survival. Antisense oligonucleotides to miR-133a are synergistic in combination with standard chemotherapy such as cisplatin in the treatment of osteosarcoma.

RELATED APPLICATIONS

This application claims benefit of priority to U.S. Provisional PatentApplication No. 61/531,942, filed Sep. 7, 2011, and U.S. ProvisionalPatent Application No. 61/696,981, filed Sep. 5, 2012.

BACKGROUND OF THE INVENTION

There is growing evidence that tumors contain a subset of cells withstem cell-like properties. These cells, often referred to either as“cancer stem cells” (CSCs) or as “tumor-initiating cells” (TICs), areresponsible for forming the bulk of tumor. These CSCs possess bothself-renewal and differentiation capabilities, and are believed to giverise to tumor heterogeneity. Furthermore, they have been shown to beassociated with the most lethal characteristics of tumors—drugresistance and metastasis. The first evidence of the existence of CSCscame from studies of hematological malignancies in 1994. More recently,CSCs have been identified in a number of solid tumors, including breast,brain, skin, lung, colon, pancreatic, liver, head and neck, prostate,ovarian, and gastric cancers.

Osteosarcoma is the most common primary bone malignancy and accounts for60% of all malignant childhood bone tumors. Before multi-agentchemotherapy, amputation provided a long-term survival rate of only˜20%. Since the 1970s, combination chemotherapy along with limb-sparingsurgery has been the main treatment for osteosarcoma. Currently, the5-year survival for patients with osteosarcoma has been reported to be50% to 80%. However, this survival rate has not improved over the last10 years, and fully 40% of osteosarcoma patients die of their disease.

Targeting molecules important in tumorigenesis, known as “targetedtherapy”, has been an exciting development in cancer treatment in thepast ten years. However, no targeted therapy is currently available forosteosarcoma. Therefore, there is a great need for developing newosteosarcoma treatments.

CD133, also known as AC133 and Prominin 1 (PROM1), is afive-transmembrane glycoprotein of unknown function. It was the firstidentified member of the prominin family of five-transmembraneglycoproteins. In 1997, Yin et al. produced a novel monoclonal antibodythat recognized the AC133 antigen, a glycosylation-dependent epitope ofCD133, and they detected expression of AC133 in CD34-positive progenitorcells from adult blood. CD133 cDNA encodes a 5-transmembrane domainmolecule with an extracellular N-terminus, a cytoplasmic C-terminus, andtwo large extracellular loops with eight consensus sites for N-linkedglycosylation. A characteristic feature of CD133 is its rapiddownregulation during cell differentiation. This feature makes CD133 aunique cell surface marker for the identification and isolation of stemcells and progenitor cells in several tissues. According to the CSCtheory, CSCs express some of the stem cell markers of normal stem cells.Therefore, tumor cells expressing CD133 independently or in combinationwith other stem cell or progenitor cell markers are thought to representCSCs. To date, however, the molecular mechanisms underlying thephenotype of CSCs expressing CD133 cell surface marker have remainedobscure.

MicroRNAs (miRNAs), first discovered in 1993 as a smallnon-protein-coding RNA, are small regulatory RNA molecules that modulatethe expression of their target genes and play important roles in avariety of physiological and pathological processes, such asdevelopment, differentiation, cell proliferation, apoptosis, and stressresponses. miRNA biogenesis requires several post-transcriptionalprocessing steps to yield the functional mature miRNA. Over the pastseveral years, many miRNAs have been investigated in various humancancers. The deregulation of the expression of miRNAs has been shown tocontribute to cancer development through various kinds of mechanisms,including deletions, amplifications, or mutations involving miRNA loci,epigenetic silencing, the dysregulation of transcription factors thattarget specific miRNAs, or the inhibition of processing. miRNAexpression profiling is of increasing importance as a useful diagnosticand prognostic tool, and many studies have indicated that miRNAs acteither as oncogenes or as tumor suppressors.

The human miRNAs miR-1 and miR-133a are located on the same chromosomalregion, in a so-called cluster. Enriched in muscle, they are miRNAs thatinhibit proliferation of progenitor cells and promote myogenesis bytargeting histone deacetylase 4 (HDAC4) and serum response factor (SRF),respectively. miR-1 has been reported to be overexpressed in individualswith coronary artery disease, while both of these miRNAs have beenreported to be expressed at low levels in cardiac hypertrophy. Despite anumber of studies, their importance in muscle physiology and diseasestill remains unclear. Recently, miR-133a (the name of which bears norelationship to the name CD133) has been considered to be dispensablefor the normal development and function of skeletal muscle. However, therelationship between these miRNAs and CSCs has, until now, been unknown.

The human miRNA miR-10b has been found to be positively associated withhigh-grade malignancy. This association held true to various types ofcancer. miR-10b is one of the most significantly upregulated miRNAs inhuman pancreatic adenocarcinomas and glioblastomas, two types of highlymetastatic and/or invasive cancers. This miRNA is highly expressed inmetastatic cancer cells propagated as cell lines, as well as inmetastatic breast tumors from patients, and is also upregulated inmetastatic hepatocellular carcinomas relative to those that are notmetastatic. The importance of miR-10b in sarcoma development has notpreviously been reported.

SUMMARY OF THE INVENTION

An aspect of the invention is a method of treating osteosarcoma. Themethod includes the step of administering to a subject in need thereofan effective amount of an antisense molecule specific for a microRNA(miRNA) selected from miR-1, miR-10b, and miR-133a.

In one embodiment, the antisense molecule is stabilized RNA.

In one embodiment, the stabilized RNA is a locked nucleic acid (LNA)oligonucleotide.

In one embodiment, the antisense molecule is DNA.

In one embodiment, the antisense molecule is 20-30 nucleotides long andcomprises a nucleotide sequence at least 90 percent identical to5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4), 5′-ACAAATTCGGTTCTACAGGGT-3′(SEQ ID NO:5), or 5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the antisense molecule is 21-30 nucleotides long andcomprises a nucleotide sequence at least 95 percent identical to5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4), 5′-ACAAATTCGGTTCTACAGGGT-3′(SEQ ID NO:5), or 5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the sequence of the antisense molecule is

-   5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4),-   5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5), or-   5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the antisense molecule is 20-30 nucleotides long andcomprises a nucleotide sequence at least 90 percent identical to

-   540 -AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the antisense molecule is 21-30 nucleotides long andcomprises a nucleotide sequence at least 95 percent identical to

-   5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the sequence of the antisense molecule is

-   5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the antisense molecule is associated with a nucleicacid delivery vehicle.

In one embodiment, the osteosarcoma is metastatic osteosarcoma.

An aspect of the invention is an isolated nucleic acid molecule 20-30nucleotides long comprising a nucleotide sequence at least 90 percentidentical to

-   5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4),-   5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5), or-   5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the isolated nucleic acid molecule is 21-30nucleotides long and comprises a nucleotide sequence at least 95 percentidentical to

-   5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4),-   5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5), or-   5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the sequence of the isolated nucleic acid molecule is

-   5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4),-   5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5), or-   5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

An aspect of the invention is an isolated nucleic acid molecule 20-30nucleotides long comprising a nucleotide sequence at least 90 percentidentical to

-   5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the isolated nucleic acid molecule is 21-30nucleotides long and comprises a nucleotide sequence at least 95 percentidentical to

-   5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the sequence of the isolated nucleic acid molecule is

-   5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7),-   5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8),-   5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9),-   5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10),-   5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11), or-   5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the nucleic acid molecule is associated with anucleic acid delivery vehicle.

An aspect of the invention is a method of assessing resistance ofosteosarcoma to an anti-cancer therapy. The method includes the stepsof:

-   obtaining a tissue sample comprising osteosarcoma cells;-   isolating from the sample cells expressing CD133;-   measuring a first level of expression by the CD133-expressing cells    of at least one microRNA (miRNA) selected from the group consisting    of miR-1, miR-10b, and miR-133a;-   contacting the CD133-expressing cells with an anti-cancer therapy;    and-   measuring a second level of expression by the CD133-expressing cells    of the at least one miRNA, wherein a second level of expression    greater than the first level of expression indicates the    osteosarcoma is resistant to the anti-cancer therapy.

In one embodiment, the anti-cancer therapy is selected from the groupconsisting of cisplatin, doxorubicin, methotrexate, and any combinationthereof.

An aspect of the invention is a method of screening for osteosarcoma.The method includes the step of performing on a tissue sample from asubject an assay specifically capable of detecting at least one microRNA(miRNA) selected from the group consisting of miR-1, miR-10b, andmiR-133a, wherein detection by the assay of the presence in the sampleof the at least one miRNA indicates the subject is at risk of havingosteosarcoma.

In one embodiment, the tissue is blood.

In one embodiment, the tissue is serum.

An aspect of the invention is a method of monitoring osteosarcoma. Themethod includes the steps of:

-   (a) performing, on a tissue sample obtained from a subject having    osteosarcoma or having been treated for osteosarcoma, an assay    specifically capable of quantifying the level of expression of at    least one microRNA (miRNA) selected from the group consisting of    miR-1, miR-10b, and miR-133a; and-   (b) repeating step (a) on a later-obtained tissue sample from the    subject, wherein a level of expression of the at least one miRNA in    the later-obtained sample greater than the level of expression of    the at least one miRNA in the earlier-obtained sample indicates the    osteosarcoma is progressive, and a level of expression of the at    least one miRNA in the later-obtained sample lesser than the level    of expression of the at least one miRNA in the earlier-obtained    sample indicates the osteosarcoma is regressive.

In one embodiment, the tissue is blood.

In one embodiment, the tissue is serum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a group of seven representative FACS analyses of variousindicated human osteosarcoma cell lines based on their expression ofCD133 (X-axis) and CD44 (Y-axis).

FIG. 2 is a collage comprising a FACS analysis depicting selection ofCD133^(high) and CD133^(low) SaOS2 cells (top panel); fourphotomicrographs depicting asymmetric cell division in the CD133^(high)population at day 1 and day 8 (middle panel); and two FACS analyses byPKH staining for each population at day 14 (bottom panel). Scale bars,50 mm.

FIG. 3 is a group of four photomicrographs and a bar graph depictingsphere-formation assays in freshly isolated CD133^(high) and CD133^(low)HOS-GFP cells. Photos were taken on day 5 and the numbers of spheres ineach well were counted (n=3 per group, **P<0.01). Scale bar, 200 μm.

FIG. 4 is a bar graph depicting drug sensitivity of CD133^(high) andCD133^(low) SaOS2 cell populations. Relative viable cells to doxorubicin(DOX, 0.03 μM), cisplatin (CDDP, 2.5 μM), and methotrexate (MTX, 0.32μM) were analyzed (n=3 per group, *P<0.05, **P<0.01).

FIG. 5 is a pair of photomicrographs and a bar graph depicting invasionassays in CD133^(high) and CD133^(low) SaOS2 cell populations (n=3 pergroup, **P<0.01). Scale bar, 200 μm.

FIG. 6 is a graph depicting quantitative polymerase chain reaction(qPCR) analysis of stem cell-associated, multiple drug-resistanttransporters and metastasis-associated genes of CD133^(high) andCD133^(low) SaOS2 cell populations. β-actin was used as an internalcontrol.

FIG. 7 is a group of eight photographic images depicting tumorigenicityof CD133^(high) and CD133^(low) HOS-luc cell populations in mice.Luminescence of the tumors xenografted with CD133^(high) (animals' rightthighs) and CD133^(low) (animals' left thighs) HOS-luc cells areidentified by in vivo imaging system (IVIS). CD133^(high) populationformed tumors with as few as 100 cells (n=5 per group).

FIG. 8 is a pair of FACS analyses depicting CD133^(high) cellpopulations in clinical osteosarcoma specimens.

FIG. 9 is a graph depicting metastasis-free survival for osteosarcomapatients based on CD133 expression. Patients with high expression ofCD133 had a median metastasis-free survival of less than 60 months(n=35, log-rank test, P=0.0262).

FIG. 10 is a Venn diagram depicting upregulated and downregulated miRNAsin CD133^(high) and CD133^(low) cells of SaOS2 and HOS.

FIG. 11 is a bar graph depicting upregulated miR-1, miR-10b, andmiR-133a in CD133^(high) populations of SaO2 and HOS cells compared toCD133^(low) population (*P<0.05, **P<0.01, ***P<0.001).

FIG. 12 is a graph depicting the expression of miR-1, miR-10b, andmiR-133a in CD133^(low) SaOS2 cells transfected with miRNAoligonucleotides compared to CD133^(low) SaOS2 cells transfected withmiR-NC (negative control) oligonucleotides (log scale, n=3 per group,*P<0.05, **P<0.01, ***P<0.001).

FIG. 13 is a graph depicting invasion assays in purified CD133^(low)cells transfected with miR-1, miR-10b, and miR-133a or NColigonucleotides (*P<0.05, **P<0.01, ***P<0.001).

FIG. 14 is a graph depicting drug resistance in CD133^(low) cellstransfected with miR-1, miR-10b, miR-133a, or miR-NC oligonucleotides(MTX, methotrexate at 0.22 mM; *P<0.05, **P<0.01).

FIG. 15 is a schematic representation of plasmid vectors utilized forstable overexpression of miR-133a in CD133^(low) cells. NruI, NotI,XbaI: restriction endonuclease sites; P_(CMV), cytomegalovirus promoter;MCS, multiple cloning site; IVS, intervening sequence; IRES, internalribosome entry site; Hyg^(r), hygromycin resistance gene; SV40 poly A,SV40 polyA tail.

FIG. 16 comprises four pairs of photographic images depictingtumorigenicity of CD133^(low) HOS-luc cells stable expressing miR-133a(right legs) compared to control CD133^(low) HOS-luc cells (left legs).Each site was injected with the indicated number of cells (10²-10⁵);luminescent evaluation was performed 90 days post injection.

FIG. 17 is a graph depicting expression of CD133 messenger RNA (mRNA) inCD133^(low) SaOS2 cells transfected with miR-1, miR-10b, and miR-133aoligonucleotides. Alteration of these miRNAs did not alter CD133expression levels. Comparison is also made to CD133^(high) cellstransfected with miR-NC (negative control) oligonucleotide (n=3 pergroup).

FIG. 18 is a graph depicting expression of miR-133a in CD133^(high)populations of freshly resected patient biopsies.

FIG. 19 is a series of photomicrographs and a related bar graphdepicting the effects of individual miRNAs, and various combinations ofthe miRNAs, on invasiveness of CD133^(low) SaOS2 cells transfected withthe indicated miRNAs. For the purposes of comparison data is alsopresented for CD133^(high) SaOS2 cells. NC, negative control.

FIG. 20 is a series of photomicrographs and related bar graph depictingthe effects of individual miRNAs, and various combinations of themiRNAs, on invasiveness of CD133^(low) MNNG/HOS cells transfected withthe indicated miRNAs. NC, negative control.

FIG. 21 is a bar graph depicting proliferation of non-transfectedCD133^(high) SaOS2 cells and CD133^(low) SaOS2 cells transfected withthe indicated miRNAs. NC, negative control. Cells were maintained inculture for 4 d prior to counting.

FIG. 22 comprises three graphs depicting (left) induction of mRNA forCD133 by doxorubicin (DOX) and cisplatin (CDDP) in 143B cells; (middle)induction of miR-1, miR-10b, and miR-133a by DOX in 143B cells; and(right) induction of miR-1, miR-10b, and miR-133a by CDDP in 143B cells(*P<0.05, **P<0.01, ***P<0.001).

FIG. 23 is a pair of juxtaposed photographic images depictingtumorigenicity of cisplatin (CDDP)-treated CD133^(low) HOS-luc cells inmice. Luminescence of the tumors xenografted with CDDP-treated cells(animals' right tibias) and saline-treated control cells (animals' lefttibias) are identified by in vivo imaging system (IVIS). CDDP-treatedCD133^(low) cells formed tumors with as few as 100 cells (n=5 pergroup).

FIG. 24 is a graph depicting knock-down of miR-1, miR-10b, and miR-133expression in CD133^(high) SaOS2 cells transfected with locked nucleicacid (LNA)-1, LNA-10b, LNA-133a, and LNA-NC oligonucleotides.

FIG. 25 is a graph depicting cell proliferation on day 4 aftertransfection of LNA-133a and LNA-NC (negative control) oligonucleotidesin CD133^(high) and CD133^(low) cells (n=3 per group; ***P<0.001).

FIG. 26 is a pair of graphs depicting (left) cell viability of indicatedcell types grown in the presence of doxorubicin (DOX, 0.4 μM, 48 h) orcisplatin (CDDP, 5 μM, 48 h) measured 24 h after transfection withLNA-133a or LNA-NC; and (right) percentage of apoptotic cells inindicated cell types grown in the presence (+) or absence (−) ofcisplatin (CDDP, 5 μM, 48 h) measured 24 h after transfection withLNA-133a or LNA-NC.

FIG. 27 is a graph depicting invasion assays in indicated LNA-treatedSaOS2 CD133^(high) and CD133^(low) populations (n=3 per group;**P<0.01).

FIG. 28 is a graph depicting quantitative polymerase chain reaction(qPCR) analysis of genes associated with steamness, drug resistance, andmetastasis of osteosarcoma in CD133^(high) cells transfected withLNA-133a and LNA-NC oligonucleotides. β-actin was used as an internalcontrol.

FIG. 29 is a schematic depicting LNA-133a (LNA) and cisplatin (CDDP)administration schedule for 143B-luc-bearing mice. IVIS, in vivo imagingsystem.

FIG. 30 is a graph depicting expression of miR-133a in 143B-luc tumorsaccording to the dose of LNA-133a (n=3 per group).

FIG. 31 is a graph depicting relative expression of miR-133a in micebearing 143B-luc tumors and treated with saline alone, LNA-133a alone,cisplatin (CDDP) alone, or LNA-133 plus CDDP (*P<0.05, ***P<0.001).

FIG. 32 is a group of four photographic images depicting macroscopicappearance, on day 36, of mice bearing 143B-luc tumors and treated withsaline alone, LNA-133a alone, cisplatin (CDDP) alone, or LNA-133 plusCDDP. Scale bar, 10 mm.

FIG. 33 is a graph depicting weight of 143B-luc tumors, on day 36, fromeach indicated treatment group (**P<0.01).

FIG. 34 is a graph depicting survival of mice bearing 143B-luc tumorsand treated with saline alone, LNA-133a alone, cisplatin (CDDP) alone,or LNA-133 plus CDDP. Kaplan-Meier analysis and log-rank test (n=5 pergroup, P=0.0013).

FIG. 35 is a schematic depicting a strategy used to identify targetgenes of miR-133a. Anti-ago2 IP, anti-Ago2 antibody immunoprecipitation.

FIG. 36 is a Venn diagram depicting candidate target messenger RNAs(mRNAs) of miR-133a according to complementary DNA (cDNA) microarray andin silico database analysis.

FIG. 37 is a graph depicting inhibition of cell growth by 10 siRNAs oncell transfection arrays in the presence of cisplatin 72 h aftertransfection (n=3 per group; NC, negative control; **P<0.01,***P<0.001).

FIG. 38 is a graph depicting invasion assay by 10 siRNAs on celltransfection arrays 72 h after transfection (n=3 per group; NC, negativecontrol; *P<0.05, **P<0.01, ***P<0.001).

FIG. 39 is a graph depicting luciferase activity in SaOS2 cellsco-transfected with miR-133a oligonucleotides and luciferase reportersfor the indicated putative miR-133a target genes.

FIG. 40 is a graph depicting inverse correlation between expression ofCD133 (CD133^(high) versus CD133^(low)) and messenger RNA (mRNA) forindicated targets of miR-133a, as measured by quantitative reversetranscriptase-polymerase chain reaction (qRT-PCR).

FIG. 41 is a graph depicting decreased expression of messenger RNA(mRNA) for the indicated miR-133a target genes in CS133^(low) SaOS2cells 48 h after transfection of miR-133a oligonucleotides compared tomiR-NC (negative control) oligonucleotides, as measured by quantitativereverse transcriptase-polymerase chain reaction (qRT-PCR).

FIG. 42 is a graph depicting increased expression of messenger RNA(mRNA) for SGMS2 in 143B-luc tumors from mice treated with LNA-133a(**P<0.01).

FIG. 43 is a series of six graphs depicting metastasis-free survival ofosteosarcoma patients sensored for miR-133a target genes SGMS2, UBA2,SNX30, DUSP11, MAST4, and ANXA2, respectively. The low expression of thedirect targets of miR-133a (except for DUSP11) were significantlycorrelated with a poor prognosis (Kaplan-Meier analysis and log-ranktest; P values as shown).

DETAILED DESCRIPTION OF THE INVENTION

Since the proposal of the cancer stem cell (CSC) hypothesis, severalstudies have been performed to identify cancer stem cells ofosteosarcoma. These cells have been detected in spherical clones underanchorage-independent, serum-starved culture conditions, as sidepopulation (SP) cells based on efflux of Hoechst 33342 dye or as CD117and stro-1 cells sorted using cell surface marker. In view of thesemodels, the inventors identified Prominin-1, the mouse homolog of humanCD133, to be highly expressed in a small fraction of osteosarcoma cells.Cells from this CD133^(high) fraction formed cluster spheres in ananchorage-independent environment, exhibited a potential forself-renewal and differentiation, expressed stem cell-associatedmarkers, and showed more invasive potential compared to the CD133^(low)fraction.

Following the characterization of the phenotype of osteosarcoma CSCs,the inventors profiled expression of several miRNAs, which distinguishcells of the CD133^(high) fraction from their more differentiatedprogeny. Among these miRNAs, miR-1, miR-10b, miR-133a were found to beupregulated in the CD133^(high) fraction compared to the CD133^(low)fraction of osteosarcoma cells. Remarkably, the inventors havediscovered these miRNAs promote chemoresistance and invasiveness ofosteosarcoma cells. These observations suggest that miR-1, miR-10b, andmiR-133a are regulators of cancer stem cells of osteosarcoma.Particularly in combination with a tailored drug delivery system, newtherapeutic agents (e.g., antisense nucleotides) targeting the miRNAsshow great promise against osteosarcoma, adding to conventionalchemotherapeutic agents, such as methotrexate, cisplatin, anddoxorubicin.

Although miRNAs are not presently used as cancer therapeutics or asvalidated targets for cancer therapeutics, successful in vivo studiessupport the notion that they could be used as innovative therapeutics toaddress unmet needs. Systemic delivery of anti-miR-10b in an orthotopicmouse model of breast cancer showed a significant reduction in thenumber and size of lung metastases, with no obvious effect on primarytumors. Ma et al. (2010) Nat Biotechnol 28:341-7. Moreover, the recentdiscovery of miRNAs as novel biomarkers in serum or plasma represents anew approach for diagnostic screening in blood. Brase et al. (2010) MolCancer 9:306. The miRNAs identified in accordance with the instantinvention also have potential as biomarkers which can be used for promptassessment of sensitivity to chemotherapeutics, early detection of localrecurrence, or distant metastasis, all of which are factors that affectthe prognosis for patients with osteosarcoma.

An aspect of the invention is a method of treating osteosarcoma. Themethod includes the step of administering to a subject in need thereofan effective amount of an antisense molecule specific for a microRNA(miRNA) selected from miR-1, miR-10b, and miR-133a. Alternatively or inaddition, the method can include the step of administering to thesubject any agent that knocks down the expression of the miRNA.

As used herein, the terms “treating” and “to treat” refers toameliorating or curing a disease or undesirable condition. For example,treating osteosarcoma refers to reducing or eliminating the burden ofosteosarcoma cells in a subject having osteosarcoma.

A “subject” as used herein refers to a mammal. In one embodiment, asubject is a human.

An effective amount of an antisense molecule specific for a microRNA isadministered to the subject in need of treatment. As used herein, an“effective amount” refers to an amount that is sufficient to achieve adesired biological outcome. For example, an effective amount to treat anosteosarcoma is an amount sufficient to reduce or eliminate thepopulation of osteosarcoma cells in a subject having osteosarcoma. Aneffective amount may vary depending on such factors as the size of thetumor, the size of the subject, the overall condition of the subject,the route of administration, the identity of the active agent, thecomposition or formulation of the active agent, and other factors wellknown in the medical and pharmaceutical arts.

Without meaning to be bound to any particular dosage, an effectiveamount can, in general, vary from 0.01 microgram (μg)/kg body weight to1000 mg/kg body weight of active agent per day when administered by aparenteral route of administration. For oral or enteral administration,an effective amount can, in general, vary from 0.1 μg/kg body weight to10,000 mg/kg body weight of active agent per day. an effective amountcan be determined, for example, based on in vitro studies and in vivoanimal studies, as well as clinical studies.

MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that areinvolved in post-transcriptional regulation of gene expression inmulticellular organisms by affecting both the stability and translationof mRNAs. miRNAs are transcribed by RNA polymerase II as part of cappedand polyadenylated primary transcripts (pri-miRNAs) that can be eitherprotein-coding or non-coding. The primary transcript is cleaved by theDrosha ribonuclease III enzyme to produce an approximately 70-ntstem-loop precursor miRNA (pre-miRNA), which is further cleaved by thecytoplasmic Dicer ribonuclease to generate the mature miRNA andantisense miRNA star (miRNA*) products. The mature miRNA is incorporatedinto an RNA-induced silencing complex (RISC), which recognizes targetmessenger RNAs (mRNAs) through imperfect base pairing with the miRNA andmost commonly results in translational inhibition or destabilization ofthe target mRNA.

The miRNA can be selected from miR-1, miR-10b, and miR-133a. In oneembodiment, the miRNA is miR-1. In one embodiment, the miRNA is miR-10b.In one embodiment, the miRNA is miR-133a. While these miRNAs have beendescribed in the art, prior to the instant invention there was norecognition or expectation that these particular miRNAs are or might beassociated with osteosarcoma, including, in particular, drug-resistantand/or aggressively invasive or metastatic phenotypes of osteosarcoma.

miR-1 has been described as a 22-nucleotide long miRNA having thesequence 5′-UGGAAUGUAAAGAAGUAUGUAU-3′ (SEQ ID NO:1).

miR-10b has been described as a 23-nucleotide long miRNA having thesequence 5′-UACCCUGUAGAACCGAAUUUGUG-3′ (SEQ ID NO:2).

miR-133a has been described as a 22-nucleotide long miRNA having thesequence 5′-UUUGGUCCCCUUCAACCAGCUG-3′ (SEQ ID NO:3).

Antisense is well described in the literature. In general, antisenseagents are nucleic acid-based molecules having a nucleotide sequencethat is complementary to the sequence of a target nucleic acid molecule,whereby association between the antisense molecule and its targetsequence molecule results in a reduced amount of expression of thetarget nucleic acid molecule.

In one embodiment the antisense molecule (anti-miRNA) is a stabilizedRNA, i.e., an RNA that, compared to naturally occurring RNA, isrelatively resistant to nuclease-mediated degradation in vitro or invivo. Numerous forms of stabilized nucleic acids, including RNA, areknown. Some stabilized RNAs include polyA 3′-terminal ends. Chemicallymodified forms of nucleic acids, including, for example and withoutlimitation, locked nucleic acids (LNAs), phosphorothioatebackbone-modified nucleic acids, and 2′-O-methyl (2′-OMe) nucleic acidshave been well described and require no further description here.Krützfeldt et al. (2005) Nature 438:685-9; Ma et al. (2010) NatBiotechnol 28:341-7.

In one embodiment, the antisense molecule is a locked nucleic acid (LNA)oligonucleotide. A locked nucleic acid nucleotide is a modifiedribonucleotide. The ribose moiety of an LNA nucleotide is modified withan extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge“locks” the ribose in the 3′-endo conformation. LNA nucleotides werefirst developed by Imanishi and colleagues and Wengel and colleagues.Obika et al. (1997) Tetrahedron Lett. 38: 8735-8; Koshkin et al. (1998)Tetrahedron 54: 3607-30.

A locked nucleic acid (LNA) oligonucleotide is a polymer of nucleotides,at least one of which is an LNA nucleotide. Any non-LNA nucleotide in anLNA oligonucleotide can be a naturally occurring or modifiedribonucleotide or deoxyribonucleotide, or an analog thereof, providedthat the LNA oligonucleotide is functional as an antisense molecule withrespect to its intended target. In one embodiment, any non-LNAnucleotide in an LNA oligonucleotide is a deoxyribonucleotide, and atleast the 3′-terminal nucleotide is an LNA nucleotide. In oneembodiment, any non-LNA nucleotide in an LNA oligonucleotide is anaturally occurring deoxyribonucleotide, and at least the two3′-terminal nucleotides are LNA nucleotides. In one embodiment, an LNAoligonucleotide is composed exclusively of LNA nucleotides.

In one embodiment, the antisense molecule is DNA.

In one embodiment, an antisense molecule specific for miR-1 comprises asequence 5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4).

In one embodiment, the sequence of an antisense molecule specific formiR-1 is 5′-ACATACTTCTTACATTCCA-3′ (SEQ ID NO:4).

In one embodiment, an antisense molecule specific for miR-10b comprisesa sequence 5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5).

In one embodiment, the sequence of an antisense molecule specific formiR-10b is 5′-ACAAATTCGGTTCTACAGGGT-3′ (SEQ ID NO:5).

In one embodiment, an antisense molecule specific for miR-133a comprisesa sequence 5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, the sequence of an antisense molecule specific formiR-133a is 5′-CAGCTGGTTGAAGGGGACCAA-3′ (SEQ ID NO:6).

In one embodiment, an antisense molecule specific for miR-1 comprises asequence 5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7).

In one embodiment, the sequence of an antisense molecule specific formiR-1 is 5′-AUACAUACUUCUUUACAUUCCA-3′ (SEQ ID NO:7).

In one embodiment, an antisense molecule specific for miR-10b comprisesa sequence 5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8).

In one embodiment, the sequence of an antisense molecule specific formiR-10b is 5′-CACAAAUUCGGUUCUACAGGGUA-3′ (SEQ ID NO:8).

In one embodiment, an antisense molecule specific for miR-133a comprisesa sequence 5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9).

In one embodiment, the sequence of an antisense molecule specific formiR-133a is 5′-CAGCUGGUUGAAGGGGACCAAA-3′ (SEQ ID NO:9).

In one embodiment, an antisense molecule specific for miR-1 comprises asequence 5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10).

In one embodiment, the sequence of an antisense molecule specific formiR-1 is 5′-ATACATACTTCTTTACATTCCA-3′ (SEQ ID NO:10).

In one embodiment, an antisense molecule specific for miR-10b comprisesa sequence 5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11).

In one embodiment, the sequence of an antisense molecule specific formiR-10b is 5′-CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO:11).

In one embodiment, an antisense molecule specific for miR-133a comprisesa sequence 5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

In one embodiment, the sequence of an antisense molecule specific formiR-133a is 5′-CAGCTGGTTGAAGGGGACCAAA-3′ (SEQ ID NO:12).

The invention further embraces antisense molecules 20 to 30 nucleotideslong comprising a contiguous sequence that is at least 90 percentidentical to any one of the foregoing antisense molecule sequences. Itis to be understood that such antisense molecules are capable ofspecifically hybridizing with or knocking down expression of the miRNAsto which they are targeted.

The invention further embraces antisense molecules 21 to 30 nucleotideslong comprising a contiguous sequence that is at least 95 percentidentical to any one of the foregoing antisense molecule sequences. Itis to be understood that such antisense molecules are capable ofspecifically hybridizing with or knocking down expression of the miRNAsto which they are targeted.

In each of the foregoing embodiments, in one embodiment the antisensemolecule includes one or more locked nucleic acid (LNA) nucleotides.Furthermore, in one embodiment the antisense molecule is composedexclusively of locked nucleic acid (LNA) nucleotides.

In one embodiment, the antisense molecule specific for a particularmicroRNA is associated with a nucleic acid delivery vehicle. As usedherein, a “nucleic acid delivery vehicle” refers to a biologicallycompatible vector useful for delivering a nucleic acid molecule to thecytoplasm of a cell. The antisense molecule can be conjugated to thenucleic acid delivery vehicle. Alternatively or in addition, theantisense molecule can be encapsulated by the nucleic acid deliveryvehicle. Examples of suitable nucleic acid delivery vehicles includeliposomes, lipids, cholesterol, hormones, and other targeting molecules.In respect of liposomes, the antisense molecule can be associated withthe outer surface of the liposome, the interior of the liposome, or boththe exterior and the interior of the liposome.

In one embodiment, the osteosarcoma is localized osteosarcoma, e.g.,osteosarcoma that is confined to one limb or one bone.

In one embodiment, the osteosarcoma is metastatic osteosarcoma.

An aspect of the invention is a method of assessing resistance ofosteosarcoma to an anti-cancer therapy. The method includes the steps of

obtaining a tissue sample comprising osteosarcoma cells;

isolating from the sample cells expressing CD133;

measuring a first level of expression by the CD133-expressing cells ofat least one microRNA (miRNA) selected from the group consisting ofmiR-1, miR-10b, and miR-133a;

contacting the CD133-expressing cells with an anti-cancer therapy; and

measuring a second level of expression by the CD133-expressing cells ofthe at least one miRNA, wherein a second level of expression greaterthan the first level of expression indicates the osteosarcoma isresistant to the anti-cancer therapy.

CD133-expressing cells can be isolated from a tissue sample using anysuitable means. For example, a cell suspension can be prepared from thetissue and then the cells can be subjected to immunochromatography with,for example, magnetic beads loaded with anti-CD133 antibody, or byfluorescence-activated cell sorting (FACS) using an appropriatelylabeled anti-CD133 antibody. Monoclonal anti-human CD133 antibodies arecommercially available from a number of suppliers.

A level of expression of a miRNA can be performed using any suitablemethod. For example, the expression level can be determined usingreverse-transcriptase polymerase chain reaction (RT-PCR) usingappropriately selected oligonucleotide primers. Alternatively or inaddition, the expression level can be determined using Northern blottingwith appropriately selected and labeled by hybridization probe.

As used herein, an “anti-cancer therapy” refers to any modality oftreatment useful to treat a cancer. Such modalities include, in general,chemotherapy, external beam radiation therapy, immunotherapy, hormonetherapy, and combinations thereof. Chemotherapeutic agents are smallmolecules (molecular weight less than about 1 kDa) are well known in themedical arts. Commonly used chemotherapeutic agents used forosteosarcoma include cisplatin (cis-diamminedichloroplatinum (II), alsoknow as CDDP, and cisplatinum, commercially available as Platinol andPlatinol-AQ), doxorubicin (also known as hydroxydaunorubicin,commercially available as Adriamycin), and methotrexate (also known asamethopterin). In one embodiment, the anti-cancer therapy is selectedfrom cisplatin, doxorubicin, methotrexate, and any combination thereof.Any two or more of these agents may be used in combination, eitherconcurrently or sequentially. In addition, any one or combination ofsuch anti-cancer therapies can be used in combination with anotheranti-cancer modality, for example, external beam radiation therapy.

In various embodiments, the osteosarcoma is identified as beingresistant to the anti-cancer therapy when the second level of expressionof at least one of miR-1, miR-10b, and miR-133a is objectively greaterthan the first level of expression. In various embodiments, theosteosarcoma is identified as being resistant to the anti-cancer therapywhen the second level of expression of at least one of miR-1, miR-10b,and miR-133a is at least 10 percent, at least 20 percent, at least 30percent, at least 40 percent, at least 50 percent, at least 60 percent,at least 70 percent, at least 80 percent, at least 90 percent, or atleast 100 percent greater than the first level of expression.

In one embodiment, the method further comprises the step of adjustingthe dose of or changing the anti-cancer therapy when the osteosarcoma isfound to be resistant to the anti-cancer therapy. For example, when theosteosarcoma is found to be resistant to the anti-cancer therapy, theanti-cancer therapy can be supplemented with or changed to anothersuitable anti-cancer therapy.

An aspect of the invention is a method of screening for osteosarcoma.The method includes the step of performing on a tissue sample obtainedfrom a subject an assay specifically capable of detecting at least onemicroRNA (miRNA) selected from the group consisting of miR-1, miR-10b,and miR-133a, wherein detection by the assay of the presence in thesample of the at least one miRNA indicates the subject is at risk ofhaving osteosarcoma. In one embodiment, the miRNA is miR-1. In oneembodiment, the miRNA is miR-10b. In one embodiment, the miRNA ismiR-133a. In one embodiment, the at least one mi-RNA is any combinationof miR-1, miR-10b, and miR-133a.

An assay specifically capable of detecting at least one microRNA (miRNA)selected from the group consisting of miR-1, miR-10b, and miR-133a canbe, for example, RT-PCR using appropriately selected oligonucleotideprimers. Alternatively or in addition, the assay can be Northernblotting with an appropriately selected and labeled hybridization probe.

In one embodiment, the tissue is blood. In one embodiment, the tissue isserum. In one embodiment, the tissue is plasma.

In one embodiment, the method further includes the step of verifying thepresence of osteosarcoma, using any suitable method, when the subject isdetermined to be at risk of having osteosarcoma. For example, verifyingthe presence of osteosarcoma can include performing a skeletal survey orspecific bone imaging analysis using X-rays or other suitable boneimaging technique, magnetic resonance imaging (MRI), computed tomography(CT), biopsy, and any combination thereof.

In one embodiment, the method further includes the step of treating thesubject for osteosarcoma.

An aspect of the invention is a method of monitoring osteosarcoma. Themethod includes the steps of

(a) performing, on a tissue sample obtained from a subject havingosteosarcoma or having been treated for osteosarcoma, an assayspecifically capable of quantifying the level of expression of at leastone microRNA (miRNA) selected from the group consisting of miR-1,miR-10b, and miR-133a; and

(b) repeating step (a) on a later-obtained tissue sample from thesubject, wherein a level of expression of the at least one miRNA in thelater-obtained sample greater than the level of expression of the atleast one miRNA in the earlier-obtained sample indicates theosteosarcoma is progressive, and a level of expression of the at leastone miRNA in the later-obtained sample lesser than the level ofexpression of the at least one miRNA in the earlier-obtained sampleindicates the osteosarcoma is regressive.

An assay specifically capable of quantifying the level of expression ofat least one microRNA (miRNA) selected from the group consisting ofmiR-1, miR-10b, and miR-133a can be, for example, RT-PCR usingappropriately selected oligonucleotide primers.

In one embodiment, the tissue is blood. In one embodiment, the tissue isserum. In one embodiment, the tissue is plasma.

In one embodiment, the method further includes the step of adjusting thedose of or changing anti-cancer therapy when the osteosarcoma is foundto be progressive. For example, when the osteosarcoma is found to beprogressive, the anti-cancer therapy can be supplemented with or changedto another suitable anti-cancer therapy.

In one embodiment, the method further includes the step of adjusting thedose of or changing anti-cancer therapy when the osteosarcoma is foundto be regressive. For example, when the osteosarcoma is found to beregressive, the anti-cancer therapy can be reduced or even suspended, orthe anti-cancer therapy can be changed to another suitable anti-cancertherapy.

The invention, now having been generally disclosed, is furtherillustrated by the following non-limiting examples.

EXAMPLES General Methods

Osteosarcoma Cell Purification From Fresh Clinical Samples. Fresh humanosteosarcoma samples ere obtained in accordance with the ethicalstandards of the institutional committee on human experimentation fromtwo patients undergoing diagnostic incisional biopsy from primary sitesof osteosarcoma before neoadjuvant chemotherapy at the National CancerCenter Hospital of Japan between October 2010 and June 2011. Thediagnosis of osteosarcoma and histologic subtypes were determined bycertified pathologists. Surgical specimens were obtained at the time ofresection and received in the laboratory within 10 minutes, immediatelymechanically disaggregated and digested with collagenase and(Nitta-gelatin) and washed in phosphate-buffered saline (PBS) twice.Single-cell suspensions were obtained by filtration through a 70 μmfilter (BD Biosciences). Cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) (Invitrogen) containing 10% heat-inactivated fetalbovine serum (FBS) (Gibco BRL) and penicillin (100 U/mL) andstreptomycin (100 μg/mL) under 5% CO₂ in a in a humidified incubator at37° C.

Cells and Cell Culture. The human osteosarcoma HuO9 cell line waspreviously established in the applicant's laboratory. The humanosteosarcoma cell lines SaOS2, U2OS, MG63, HOS, MNNG/HOS, and 143B werepurchased from the American Type Culture Collection (ATCC). Thetransformed embryonic kidney cell line 293 was also obtained from theATCC. SaOS2 and HuO9 cells were cultured in RPMI 1640 medium (GibcoBRL). U2OS, MG63, HOS, MNNG/HOS, 143B, and 293 cells were cultured inDMEM (Invitrogen). All media were supplemented with 10% heat-inactivatedFBS (Gibco BRL) and penicillin (100 U/mL) and streptomycin (100 μg/mL).The cells were maintained under 5% CO2 in a humidified incubator at 37°C.

Cell Sorting and Flow Cytometry. Cell sorting by flow cytometry wasperformed on osteosarcoma cell lines and clinical samples usingphycoerythrin (PE)-conjugated monoclonal mouse anti-human CD 133/2(293C3, Miltenyi Biotec) and allophycocyanin (APC)-conjugated monoclonalmouse anti-human CD44 (eBioscience). Isotype control mouse IgG1κ-PE(eBioscience) served as a control. Samples were analyzed and sorted onthe JSAN cell sorter (Baybioscience) and the BD FACS Ariall (BDBiosciences). Viability was assessed using trypan blue exclusion.Results were analyzed with FlowJo software (Tree Star).

Cell Proliferation and Cytotoxity Assay. Cell proliferation rates andcell viability as an indicator for the relative sensitivity of the cellsto doxorubicin, cisplatin, and methotrexate were determined usingTetraColor ONE Cell proliferation Assay System (Seikagaku) according tothe manufacturer's instructions. Cells growing in the logarithmic phasewere seeded in 96-well plates (5×10³/well), allowed to attach overnight,and then were treated with varying doses of doxorubicin (Sigma),cisplatin (Alexis), and methotrexate (Sigma) for 72 h. Triplicate cellswere used for each treatment group. Absorbance was measured at 450 nmwith a reference wavelength at 650 nm on EnVision (Wallac). The relativenumber of viable cells was expressed as the percent of cell viability.

Sphere Formation. Osteosarcoma cells were plated at 5,000-10,000cells/well in 300 μL of serum-free DMEM/F12 medium (Invitrogen),supplemented with 20 ng/mL human recombinant epidermal growth factor(EGF) (Sigma-Aldrich), 10 ng/mL human recombinant basic fibroblastgrowth factor (bFGF) (Invitrogen), 4 μg/mL insulin (Sigma-Aldrich),B-27® (1:50; Invitrogen), 500 units/mL penicillin (Invitrogen, and 500μg/mL streptomycin (Invitrogen). Cells were cultured in suspension in24-well ultra-low attachment plates (Corning). Cells were replenishedwith 30 μL of supplemented medium every second day. Spheres were countedon day 5 in triplicate wells. Cell culture was carried out at 37° C. ina 5% CO₂ humidified incubator.

Invasion Assay. Invasion assays were performed by using 24-well BDBioCoat Invasion Chambers with Matrigel (Becton-Dickinson). 1×10⁵ cells,suspended in 500 μL DMEM or RPMI 1640 medium without FBS, were added tothe upper chamber, and DMEM or RPMI 1640 medium with 10% FBS was addedto the lower chamber. After incubation for 24 h or 36 h, the cells onthe upper surface of the filter were completely removed by wiping themwith cotton swabs. The filters were fixed in methanol and stained with1% toluidine blue in 1% sodium tetraborate (Sysmex). Filters were thenmounted onto slides, and cells on lower surfaces were counted. Eachassay was performed in triplicate.

miRNA Profiling. miRNA expression profiling was performed using miRNAmicroarrays manufactured by Agilent Technologies (Santa Clara, Calif.),each containing 866 human miRNAs (Agilent Technologies[http://www.chem.agilent.com/scripts/PHome.asp]). Three independentlyextracted RNA samples of CD133^(high) and CD133^(low) cells just afterisolation were used for array analyses in each cell line. Labeling andhybridization of total RNA samples were performed according to themanufacturer'protocol. Microarray results were extracted using AgilentFeature Extraction software (v10.7.3.1) and analyzed using GeneSpring GX11.0.2 software (Agilent Technologies).

Clinical Samples for Survival Correlation Studies of CD133, miR-133a,and Targets of miR-133a. Osteosarcoma tissue samples were obtained bydiagnostic incisional biopsy from primary sites of osteosarcoma beforeneoadjuvant chemotherapy at the National Cancer Center Hospital of Japanbetween June 1997 and September 2010. Patients older than 40 y andhaving primary tumors located outside the extremities were excluded.Each fresh tumor sample was cut into two pieces, one of which wasimmediately cryopreserved in liquid nitrogen, and the other fixed withformalin. The diagnosis of osteosarcoma and histologic subtypes weredetermined by certified pathologists. Only osteosarcoma samples with theosteoblastic, chondroblastic, fibroblastic, and telangiectatichistologic subtypes were included. The response to chemotherapy wasclassified as good if the extent of tumor necrosis was 90% or greater.For the survival correlation studies of CD133 and the targets ofmiR-133a, available 35 cDNA samples from cDNA library were used, whileRNA from available 48 formalin-fixed paraffin-embedded (FFPE) sampleswere used for the correlation study of miR-133a. The clinicalinformation of the patients is included in Tables 7 and 10 (below). Allpatients provided written informed consent authorizing the collectionand use of their samples for research purposes. The study protocol forobtaining clinical information and collecting samples was approved bythe Institutional Review Board of the National Cancer Center of Japan.

RNA Isolation and Quantitative Real-Time RT-PCR of mRNAs and miRNAs.Total RNA was purified from cells and tumor tissues with an RNeasy MiniKit and RNase-Free DNase Set (QIAGEN). For quantitative polymerase chainreaction (qPCR) of mRNAs, cDNA was synthesized using High-Capacity cDNAReverse Transcription Kit (Applied Biosystems). For each qPCR reaction,equal amounts of cDNA were mixed with Platinum SYBER Green qPCR SuperMix(Invitrogen) and specific primers (Table 1). Gene expression levels werenormalized by beta actin (ACTB) or glyceraldehyde 3-phosphatedehydrogenase (GAPDH). For qPCR of miRNAs, miRNA was converted to cDNAusing the TaqMan MicroRNA Reverse Transcription Kit (AppliedBiosystems). RNU6B small nuclear RNA was amplified as an internalcontrol. qPCR was performed using each miRNA-specific probe includedwith the TaqMan MicroRNA Assay on a Real-Time PCR System 7300 and SDSsoftware (Applied Biosystems).

TABLE 1 Sequences of primers for real-time RT-PCR analysis. SEQ For/ IDGene Rev Sequence (5′-Sequence-3′) NO: CD133 For GGACCCATTGGCATTCTC 13Rev CAGGACACAGCATAGAATAATC 14 Oct3/4 For AGTGAGAGGCAACCTGGAGA 15 RevACACTCGGACCACATCCTTC 16 Nanog For CAGTCTGGACACTGGCTGAA 17 RevCTCGCTGATTAGGCTCCAAC 18 Sox2 For TGGTACGGTAGGAGCTTTGC 19 RevTTTTTCGTCGCTTGGAGACT 20 ABCB1 For CATGCTCCCAGGCTGTTTAT 21 RevGTAACTTGGCAGTTTCAGTG 22 AGCG2 For TGCAACATGTACTGGCGAAGA 23 RevTCTTCCACAAGCCCCAGG 24 ABCC2 For ACAGAGGCTGGTGGCAACC 25 RevACCATTACCTTGTCACTGTCCATGA 26 ezrin For CGGGACAAGTACAAGGCACTGCGGCAGATCCGG27 Rev CCGGATCTGCCGCAGTGCCTTGTACTTCCG 28 β4- ForTGACGATCTGGACAACCTCAAGCA 29 Integrin Rev ATCCAATGGTGTAGTCGCTGGTGA 30MMP13 For GATACGTTCTTACAGAAGGC 31 Rev ACCCATCTGGCAAAATAAAC 32 CXCR4 ForGGAGGGGATCAGTATATACA 33 Rev GAAGATGATGGAGTAGATGG 34 SGMS2 ForCAATTCCTTGCTGCTTCTCC 35 Rev GCCTTTGTTTTGCTCCTCAG 36 UBA2 ForAAAAAGGGTGTGACCGAGTG 37 Rev GCATCTTCTTCCCCAAACAA 38 SNX30 ForCCTGAACGCCTACAAGAAGC 39 Rev ATGGTTCCCAGTTTGAGTGC 40 DOLPP1 ForGAGAGGAGTGAGGCAACAGG 41 Rev ACCCCAGACACAGGTTTGAG 42 DUSP11 ForGAGACGCGACTTTTCAGGAC 43 Rev GATCCAAAGGGGAAAAGCAT 44 CUL4B ForGTTCTGGCGAAAAATCCAAA 45 Rev TCGAACAATTGCAGCATCA 46 ROD1 ForCATTCCTGGGGCTAGTGGTA 47 Rev CCATCTGAACCAAGGCATTT 48 ZNF701 ForATCCCGTGGAGTGAAGGTC 49 Rev TCTCCAGCATCACGTCTCTG 50 MAST4 ForAGCCCATTTTTCATTTGCAC 51 Rev TCGTCTGGTGTTGGTTGGTA 52 ANXA2 ForCCTGAGCGTCCAGAAATGG 53 Rev GGACTGTTATTCGCAAGCTGGTT 54 ACTB ForCATGAAGTGTGACGTGGACA 55 Rev CACGGAGTACTTGCGCTCAG 56 GAPDH ForGACTTCAACAGCGACACCC 57 Rev GCCAAATTCGTTGTCATACCA 58

Transfection with Synthetic miRNAs, LNAs, and siRNAs. Synthetic hsa-miRs(Pre-miR-hsa-miR-1, -10b, -133a, and negative control (NC; AppliedBiosystems, Table 2) and locked nucleic acids (LNAs) (LNA-1, -10b,-133a, and negative control, Exiqon, Table 3) were transfected into eachtype of cells at 30 nM each (final concentration) using DharmaFECT I (GEHealthcare). Synthetic siRNAs (Bonac corporation, Table 4) weretransfected into each type of cells at 100 nM each (final concentration)using DharmaFECT I (GE Healthcare). After 24 hours of incubation, cellswere treated with chemotherapeutic agents for cytotoxicity assay orreseeded into invasion chambers for invasion assay.

TABLE 2 Sequences of miRNA products SEQ Sense/ ID miRNA AntisenseSequence (5′-Sequence-3′) NO: hsa-miR-1-2 Sense UGGAAUGUAAAGAAGUAUUGUAU1 Antisense UACAUACUUCUUAUGUACCC 59 hsa-miR-10b SenseUACCCUGUAGAACCGAAUUUGUG 2 Antisense ACAGAUUCGAUUCUAGGGGAAU 60 hsa-miR-Sense UUUGGUCCCCUUCAACCAGCUG 3 133a-1 Antisense AGCUGGUAAAAUGGAACCAAAU61

TABLE 3 Sequences of LNA products miRNA Sequence (5′-Sequence-3′)SEQ ID NO: hsa-miR-1-2 ACATACTTCTTTACATTCCA 10 hsa-miR-10bACAAATTCGGTTCTACAGGGT 11 hsa-miR-133a-1 CAGCTGGTTGAAGGGGACCAA 12

TABLE 4 Sequences of siRNAs SEQ Sense/ ID Gene AntisenseSequence (5′-Sequence-3′) NO: SGMS2 Sense CCACUAGAGUGGUGGAAAAdTdT 62Antisense UUUUCCACCACUCUAGUGGdTdT 63 UBA2 Sense GGACUGGGCUGAAGUACAAdTdT64 Antisense UUGAUCUUCAGCCCAGUCCdTdT 65 SNX30 SenseCCGAGAAGUUUGUGGUAAAdTdT 66 Antisense UUUACCACAAACUUCUCGGdTdT 67 DOLPPSense CUUCCUAAUCCGAGACACAdTdT 68 Antisense UGUGUCUCGGAUUAGGAAGdTdT 69DUSP11 Sense CCAGAGGAUUUGCCAGAAAdTdT 70 AntisenseUGUGUCUCGGAUUAGGAAGdTdT 71 CUL4B Sense GGUGAACACUUAACAGCAAdTdT 72Antisense UUGCUGUUAAGUGUUCACCdTdT 73 ROD1 Sense GGGAAUGACAGCAAGAAAUdTdT74 Antisense AUUUCUUGCUGUCAUUCCCdTdT 75 ZNF701 SenseCCAUAAUGAAGGAGGUCUUdTdT 76 Antisense AAGACCUCCUUCAUUAUGGdTdT 77 ANXA2Sense UGACCAAGAUGCUCGGGAUdTdT 78 Antisense AUCCCGAGCAUCUUGGUCAdTdT 79MAST4 Sense GGAGGUACCUUCUUCCAAAdTdT 80 Antisense UAUCAAACUUCCUCUUCUGdTdT81

Establishment of miR-133a Stably Expressing Cell Line. miR-133a vectorswere constructed by inserting cloning sequences including thefull-length of the mature microRNA sequences into the pIRES-hyg vector(Clontech). The microRNA and control vectors were transfected intofreshly isolated osteosarcoma CD133^(low) HOS cells by calcium phosphateco-precipitation. The transfectants were split and grown in selectivemedium with 200 μg/mL of hygromycin. Hygromycin-resistant colonies werechosen and expanded in medium containing 200 μg/mL of hygromycin. Thesequences of miR-133a constructs were confirmed by DNA sequencing (ABI3130 sequencer, Applied Biosystems), and microRNA overexpression wasconfirmed by qRT-PCR. RNU6B served as the endogenous control.

Tumor Transplantation Experiments. Animal experiments were performed incompliance with the guidelines of the Institute for Laboratory AnimalResearch, National Cancer Center Research Institute. Athymic nude miceor NOD/SCID mice (CLEA Japan) were purchased at 4 weeks of age and givenat least 1 week to adapt to their new environment prior to tumortransplantation. On day 0, mice were anesthetized with 3% isoflurane andthe right leg disinfected with 70% ethanol. Cells were aspirated into a1 mL tuberculin syringe fitted with a 27-G needle. The needle wasinserted through the cortex of the anterior tuberosity of the tibia witha rotating movement to avoid cortical fracture. Once the bone wastraversed, the needle was inserted further to fracture the posteriorcortex of the tibia. A 100 μL volume of solution containing freshlyisolated CD133^(high) and CD133^(low) HOS-Luc (10², 10³, 10⁴, 10⁵ cellsper site ) or 143B-Luc (1.5×10⁶) was injected while slowly moving backthe needle.

Monitoring Tumor Growth, Lung Metastasis, and Toxicity with/withoutLNA-Anti-miR-133a. For the assessment of tumorigenicity betweenCD133^(high) and CD133^(low) HOS-Luc cells, NOD/SCID mice were injectedwith D-luciferin (150 mg/kg, Promega) by intraperitoneal injection. Tenminutes later, photons from firefly luciferase were counted using theIVIS imaging system (Xenogen Corp.) according to the manufacturer'sinstructions. Each experimental condition included 5 animals per groupand monitoring once a week. For the evaluation of LNA-anti-miR-133aadministration into spontaneous lung metastasis of osteosarcoma modelmice, individual mice were injected with 10 mg/kg of LNA-anti-miR-133aor saline via the tail vain. LNA were injected on following days 4, 11,18, 25, 32 postinoculation of 143B-Luc cells. each experimentalcondition included 10 animals per group. The development of subsequentlung metastasis was monitored once a week in vivo by the bioluminescentimaging described above for 5 weeks. All data were analyzed usingLivingImage software (version 2.50, Xenogen). On day 36, the primarytumor and lung in 5 mice of each group were resected at necropsy fortheir weight, bioluminescent, and histological analyses. The bloodexamination, weight of whole body as well as heart, liver, and skeletalmuscle, and histopathological examination were performed for theassessment of toxicity. The remaining mice were observed for theirsurvival period.

Comprehensive Collection and Identification of miR-133a Target mRNAs. Tocollect comprehensive downstream targets of miR-133a, cDNA microarrayprofiling from two experimental approaches were performed. First,candidate genes were collected from cDNA microarray analysis performedfrom collected total RNA of SaOS2 CD133^(low) cells transduced withmiR-133a or negative control (NC). Second, cDNA microarray analysis wasperformed from collected total RNA from anti-Ago2 antibodyimmunoprecipitation (Ago2-IP) in CD133^(low) cells transduced withmiR-133a or NC. Downregulated genes in the former method with 1.5 folddecrease and upregulated genes in the latter method with 2.0 foldincrease were defined as candidates by reference to in silico databasesTargetScanHuman 6.0 (http://www.targetscan.org/).

Luciferase Reporter Assays. Each fragment of 3′UTR of SGMS2 (nt1656-1879 (binding site) of NM_152621), UBA2 (nt 2527-2654 (bindingsite) of NM_005499), DUSP11 (nt 1180-1572 (binding site) of NM_003584),MAST4 (nt 8017-8096 (binding site) of NM_001164664), SNX30 (nt 6659-7611(binding site) of NM_1101012944) and CDS of ANXA2 (nt 244-743 (bindingsite) of NM_001002857) were amplified and cloned into the XhoI and NotIsites of firefly and renilla luciferase reporter genes of a psiCHECK-2Vector (Promega). All PCR products cloned into the plasmid were verifiedby DNA sequencing to ensure that they were free of mutations and in thecorrect cloning direction. Primer sequences are listed in Table 5. Forthe luciferase reporter assay, HOS cells were co-transfected with 100 ngof luciferase constructs and 100 nM synthetic miR-133a molecules orcontrol (non-targeting siRNA oligonucleotide, Qiagen). Firefly andrenilla luciferase activities were measured using the Dual-LuciferaseReporter Assay (Promega) 48 h after transfection. Results were expressedas relative renilla luciferase activity (renilla luciferase/fireflyluciferase).

TABLE 5 Sequences of primers for luciferase reporter assays SEQ For/ IDGene Rev Sequence (5′-Sequence-3′) NO: SGMS2- ForGCTCGAGTAAAGCAAAACAAAGGCATCAGC 82 UTR Rev GCGGCCGCAAGGCTTGTCACCAATGAATGA83 SGMS2mu- For AAATGTCAACCATTTTGTGTAAACGATTA 84 UTR RevAAATGGTTGACATTTCTTCATTTACCAG 85 UBA2-UTR ForGCTCGAGTAATACCGCCTCGTATGTCTGTG 86 Rev GCGGCCGCAATGCAGATGCCATTTATTTGGT 87UBA2mu- For TTATGTCAACCATAAATGGCATCTGCATT 88 UTR RevTTATGGTTGACATAAGTATAGTCGTTAT 89 SNX30-1- ForGCTCGAGTAACCCTGTTGGACAGGATTGAT 90 UTR RevGCGGCCGCAATTTTTAAAGAAAGCATCTTTTA 91 TGG SNX30mu- ForTCTTATCAACCCACTTCAGTCAGAAATGT 92 1-UTR Rev AGTGGGTTGATAAGACTGCGAACAATCA93 DUSP11- For GCTCGAGTAAAAACCTGTCCTGGAATTCTACC 94 UTR RevGCGGCCGCAAGATGGCCTTTGGGTTCAATAA 95 DUSP11mu- ForCTGGATCAACGAGCTGGCCTGAAAATTAC 96 UTR Rev GCTCGTTGATCCAGGTAGAATTCCAGGA 97MAST4-UTR For GCTCGAGTAACTCCCCCAGCTAGGAAACAG 98 RevGCGGCCGCAAAGAGATGGGGCGGTCAGT 99 MAST4mu- ForGACGTTCAACCGCCATCCCCAGCCCCAAA 100 UTR Rev TGGCGGTTGAACGTCTCTGCCCACGTTC101 ANXA2-UTR For GCTCGAGTAAGCGGGATGCTTTGAACATT 102 RevGCGGCCGCAACTCCAGCGTCATAGAGATCC 103 ANXA2mu- ForATCAATCAACCAGGTGTGGATGAGGTCAC 104 UTR Rev ACCTGGTTGATTGATGGCTGTTTCAATG105

Immunohistochemistry. For the staining of CD133 and targets of miR-133a,slides of osteosarcoma clinical samples and xenografted tumors wereprepared. Endogenous peroxidase was inhibited with 1% H₂O₂ (30 min).Slides were heated for antigen retrieval in 10 mM sodium citrate (pH5.0). Subsequently, the slides were incubated with monoclonal mouseanti-human CD133/2 (1:10 dilution, Miltenyi Biotec), monoclonal mouseanti-human SGMS2 (1:50 dilution, Abcam), or isotype-matched controlantibodies overnight at 4° C. Immunodetection was performed usingImmPRESS peroxidase polymer detection reagents (Vector Laboratories) andMetal Enhanced DAB Substrate Kit (Thermo Fisher Scientific) according tothe manufacturer's instructions. Staining was revealed bycounter-staining with hematoxylin.

Statistical Analysis. All statistical analyses were performed using SPSSsoftware (SPSS, Inc.; Chicago, Ill.), with the exception of thesignificance in bar graphs, in which case analyses were performed byapplying the Student's t-test. Differences in the CD133 expressionamount different clinicopathological data were analyzed by Chi-square(χ²) test. Cases with ΔCt lower than the mean value were classified ashaving high CD133 expression, while cases with ΔCt higher than the meanvalue were classified as having low CD133 expression. The Kaplan-Meiermethod and the log-rank test were used to compare the survival ofpatients with CD133^(high) and CD133^(low) primary tumors. Survivalperiod was defined as the time from diagnosis until death whereas livingpatients were censored at the time of their last follow-up. For thecalculation of differences in the expressions of miR-133a and itstargets, the same procedure was applied. In all these analyses, a Pvalue of 0.05 or less was considered to be a significant difference.

Example 1 A Small Subset of Cells of Osteosarcoma Cell Line ExpressesCD133

Osteosarcoma cell lines SaOS2, HOS, U2OS, MNNG/HOS, MG63, 143B, and HuO9were screened for markers of mesenchymal stem cells or neural stem cellsthat have been considered as the origin of sarcoma. Basu-Roy, U et al.(2011) Oncogene 31:2270-82; Kuhn, N Z et al. (2010) J. Cell. Physiol.222:268-77. As a result, CD133, the structural homolog of prominin-1,was found in all cell lines at a small population ranging from 0.04% to8.47%, whereas CD44 was found in a large population (FIG. 1). SaOS2,MNNG/HOS, and HOS were found to be particularly strong in theirexpression of CD133 (8.47, 8.13, and 7.69 percent, respectively).

Single-cell proliferation of freshly isolated cell population wasobserved using PKH dye, which is a fluorescent dye that binds to cellmembranes and segregates in daughter cells after each cell division.Normally, PKH concentration decreases with each cell division, so thatquiescent cells remain PKH^(high) and dividing cells becomeprogressively PKH^(low). Moreover, normally PKH7 dye is distributedequally between daughter cells, whereas rapidly dividing cells, e.g.,cancer cells, exhibit asymmetric division.

The CD133^(high) cell population generated both CD133^(high) andCD133^(low) populations with different proliferative fates: one that isquiescent (PKH^(high)) and another that divides actively (PKH^(low)). Asingle PKH26^(high) cell of CD133^(high) fraction showed asymmetricdivision; a small number of PKH26^(high) cells, presenting as dormantcells, were observed surrounded by PKH26^(low) cells on day 8, whichwere identified as a fraction with both CD133^(high) and CD133^(low)cells on FACS analysis. On the other hand, a single PKH67^(high) cell ofCD133^(low) SaOS2 fraction showed symmetric division; a colony withPKH67^(low) cells was observed, which was identified as a CD133^(low)fraction in FACS analysis two weeks after isolation (FIG. 2). Nodifference in cell division according to the expression of CD44 wasobserved.

Further examinations were performed to identify other phenotypes ofCD133^(high) and CD133^(low) population. A total of 5×10³ CD133^(high)and CD133^(low) cells were sorted and cultured immediately underconditions of serum-free, growth factor-supplementedanchorage-independent environment. Within two weeks of culture, moreosteosarcoma spheres were observed from CD133^(high) cells thanCD133^(low) cells (FIG. 3).

Example 2 CD133^(high) Cells Exhibit Increased Drug Resistance,Invasiveness, and Tumorigenesis

Since drug resistance is one of the important properties of TICs,populations of CD133^(high) and CD133^(low) cells were observed in thetreatment condition with doxorubicin (DOX), cisplatin (CDDP), andmethotrexate (MTX), which are standard chemotherapeutics againstosteosarcoma. CD133^(high) cells were more resistant to thesechemotherapeutics than CD133^(low) cells (FIG. 4). Furthermore,CD133^(high) cells exhibited higher capability of invasion thanCD133^(low) cells (FIG. 5). qRT-PCR of mRNA from freshly isolatedCD133^(high) and CD133^(low) cells revealed that CD133^(high) SaOS2cells expressed enhanced levels of Oct3/4, Nanog, and Sox-2, which aretranscription factors that play a critical role in maintenance ofself-renewal and pluripotency of embryonic stem cells as well as CSCs orTICs (Levings, P P et al. (2009) Cancer Res. 69:5648-55; Basu-Roy U etal. (2011) Oncogene 31:2270-82); multidrug resistance transporter genesABCB1, ABCC2, ABCG2; and metastasis-associated genes β4-integrin, ezrin,MMP-13, and CXCR4 (Tang, N et al. (2008) Clin. Orthop. Relat. Res.466:2114-30; Osaki, M et al. (2011) Mol. Ther. 19:1123-30) (FIG. 6).Most importantly, the CD133^(high) HOS fraction showed strongertumorigenicity in vivo than the CD133^(low) HOS fraction; CD133^(high)cells could form tumors from as few as 100 cells, whereas CD133^(low)cells could not (FIG. 7). Results are also shown in Table 6.

TABLE 6 Tumor development in vivo using osteosarcoma CD133^(high) andCD133^(low) populations alone Cell Type Tumor Incidence Cell NumberCD133^(high) 5/5 100,000 5/5 10,000 5/5 1,000 4/5 100 CD133^(low) 4/5100,000 1/5 10,000 1/5 1,000 1/5 100

High-Level Expression of CD133 Messenger RNA is a Marker for PoorSurvival Rates of Osteosarcoma Patients

To evaluate the clinical importance of CD133, cell lines establishedfrom fresh human osteosarcoma biopsies were analyzed by flow cytometerand found to contain CD133^(high) population at a rare frequency <10%(FIG. 8). Furthermore, a clinical study of 35 osteosarcoma patientsrevealed that high expression levels of CD133 messenger RNA (mRNA)correlated with significantly worse overall survival rates ofosteosarcoma patients (log-rank test, P=0.0262). Results are shown inFIG. 9 and Table 7.

TABLE 7 Uni- and multivariate analyses and the relationship betweenclinicopathologic variables and CD133 expression in 35 cases NumberCorrelation of CD133 CD133 (CD133) x² Variable cases Low High (P value)Age (years) 0.120 0-10 7 6 1 11-20 25 11 14 21+ 3 1 2 Gender 0.164 Male23 14 9 Female 12 4 8 Site 0.319 Femur 21 12 9 Tibia 9 5 4 Humerus 2 1 1Other 3 0 3 Histology 0.394 Osteoblastic 16 9 7 Chondroblastic 6 4 2Fibroblastic 2 0 2 Other, NA* 11 5 6 Metastasis at diagnosis 0.045Present 4 0 4 Absent 31 18 13 Neoadjuvant chemotherapy 0.425 MTX +DOX/CDDP 21 10 11 IFO + DOX/CDDP 13 8 5 Other 1 0 1 Response toneoadjuvant 0.088 chemotherapy Good (necrosis > 90%) 11 6 5 Poor(necrosis < 90%) 20 12 8 NA* 4 0 4 CD133 mRNA expression High 17 0 17Low 18 18 0

Example 4 miR-1, miR-10b, and miR-133a are Upregulated in CD133^(high)Cells Compared to CD133^(low) Cells

miRNA expression profiling has been reported to be a useful diagnosticand prognostic tool, and many studies have indicated that certain miRNAsact as either an oncogene or a tumor suppressor. Croce, C M (1009) Nat.Rev. Genet. 10:704-10. In order further to characterize the molecularmechanism underlying the CD133^(high) and CD133^(low) phenotypes, miRNAprofiling of isolated CD133^(high) and CD133^(low) osteosarcoma SaOS2and HOS cells was performed using microarray analysis containing 866sequence-validated human miRNAs. Results, shown in FIG. 10 and Table 8,revealed that 3 miRNAs were upregulated at >2-fold changes inCD133^(high) cells compared to CD133^(low) cells. A second round of qPCRvalidation study revealed miR-1, miR-10b, and miR-133a were consistentwith the microarray data (FIG. 11).

TABLE 8 microRNA expression profile of CD133^(high) versus CD133^(low)osteosarcoma cells Fold change Fold change (SaOS2 CD133^(high) (HOSCD133_(high) miRNA vs CD133^(low)) Regulation vs CD133^(low)) RegulationCommonly upregulated miRNAs in SaOS2 and HOS with >2 fold change inCD133^(high) cells compared to CD133^(low) cells hsa-miR-1 7.23 up 3.81up hsa-miR-500* 5.39 up 3.99 up hsa-miR-660 2.09 up 2.05 up UpregulatedmiRNAs in SaOS2 with >2 fold change in CD133^(high) cells compared toCD133^(low) cells hsa-miR-551b 9.49 up hsa-miR-30c 9.19 up hsa-miR-148b8.26 up hsa-miR-193a-3p 7.77 up hsa-miR-1 7.23 up hsa-miR-221* 6.76 uphsa-miR-24-1* 6.36 up hsa-miR-1825 5.78 up hsa-miR-500* 5.39 uphsa-miR-92a-2* 4.36 up hsa-miR-1202 3.39 up hsa-miR-424 3.23 uphsa-miR-19b-2* 2.87 up hsa-miR-29c 2.42 up hsa-miR-494 2.37 uphsa-miR-10b 2.16 up hsa-miR-374a 2.11 up hsa-miR-660 2.09 uphsa-miR-30c* 2.03 up Downregulated miRNAs in SaOS2with <2 fold change inCD133^(high) cells compared to CD133^(low) cells hsa-miR-1281 9.45 downhsa-miR-195 6.56 down hsa-miR-129-5p 5.74 down hsa-miR-129-3p 4.98 downhsa-miR-183 4.89 down hsa-miR-1305 4.76 down hsa-miR-1275 4.65 downhsa-miR-484 4.55 down hsa-miR-1268 4.51 down hsa-miR-186 4.51 downhsa-miR-181a* 4.46 down hsa-miR-744* 2.72 down hsa-miR-96 2.65 downhsa-miR-142-3p 2.35 down hcmv-miR-US25-2-5p 2.31 down Upregulated miRNAsin HOS with >2 fold change in CD133^(high) cells compared to CD133^(low)cells hsa-miR-1181 12.78 up hsa-miR-133b 7.22 up hsa-miR-532-5p 7.10 uphsa-miR-338-3p 6.30 up hsa-miR-9 5.95 up hsa-miR-34c-5p 5.37 uphsa-miR-378* 5.26 up hsa-miR-181a* 5.04 up hsa-miR-145* 4.97 uphsa-miR-1271 4.97 up hsa-miR-362-3p 4.67 up hsa-miR-152 4.63 uphsa-miR-663 4.46 up hsa-miR-9* 4.15 up hsa-miR-340 4.10 up hsa-miR-7444.07 up hsa-miR-500* 3.99 up hsa-miR-1 3.81 up hsa-miR-1305 3.33 uphsa-miR-744* 3.11 up hsa-miR-629 2.88 up hsa-miR-629* 2.71 uphsa-miR-145 2.55 up hsa-miR-1246 2.47 up hsa-miR-21* 2.39 uphsa-miR-450a 2.35 up hsa-miR-425* 2.31 up hsa-miR-148a 2.30 uphsa-let-7f-1* 2.26 up hsa-miR-301b 2.21 up hsa-miR-1826 2.15 uphsa-miR-128 2.15 up hsa-miR-378 2.14 up hsa-miR-126 2.13 up hsa-miR-5982.06 up hsa-miR-1915 2.05 up hsa-miR-660 2.05 up hsa-miR-933 2.02 upDownregulated miRNAs in HOS with <2 fold change in CD133^(high) cellscompared to CD133^(low) cells hsv1-miR-H6 4.60 down hsa-miR-1539 4.02down hsa-miR-483-3p 3.76 down hsa-miR-328 3.72 down hsa-miR-132* 3.67down hsa-miR-129* 3.66 down hsa-miR-548c-5p 3.13 down hsa-miR-1825 2.05downhsa, Homo sapiens.

miRNA and miRNA* are the two strands of the double-stranded RNA productof dicer processing of the stem loop precursor miRNA. miRNA is the“guide” strand that eventually enters RISC, and miRNA* is the other“passenger” strand. The level of miRNA* present in the cell is low (≦15%relative to the corresponding miRNA). In cases where there is a higherproportion of passenger strand present in the cell, the nomenclaturemiRNA-3p/miRNA-5p is used instead of miRNA/miRNA*. miRNA-3p is the miRNAderived from the 3′ arm of the precursor miRNA, and miRNA-5p is themiRNA derived from the 5′ arm of the precursor miRNA.

Example 5 Transfection of CD133^(low) Cells with miR-133 ConfersProperties Associated with CD133^(high) Cells, and High Expression ofmiR-133a is Correlated with Poor Clinical Prognosis

To determine whether these miRNAs can inhibit these phenotypes ofosteosarcoma tumor-initiating cells, expression levels of miR-1,miR-10b, and miR-133a were manipulated in CD133^(low) cells (FIG. 12).These miRNAs, especially miR-133a, enhanced the invasiveness ofCD133^(low) cells compared to miR-NC (negative control) oligonucleotides(FIG. 13). Interestingly, transfection of all these miRNAs dramaticallyenhanced the invasion of CD133^(low) cells (FIG. 13). Cell proliferationand drug resistance were slightly enhanced in CD133^(low) cells bymiR-133 transfection (FIG. 14). Stable overexpressing miR-133a HOSCD133^(low) cells (FIG. 15) showed strong (>2-fold) ability to formshapes than control CD133^(low) cells under anchorage serum-freeenvironment and could develop tumors with as few as 100 cells in vivowhereas control CD133^(low) cells could not (FIG. 16 and Table 9).

TABLE 9 Tumor development in vivo using osteosarcoma CD133^(low)populations stably overexpressing miR-133a Call Type Tumor IncidenceCell Number CD133^(low)miR-133a 5/5 100,00 4/4 10,000 5/5 1,000 2/5 100CD133^(low)EV¹ 5/5 100,000 1/4 10,000 0/5 1,000 0/5 100 CD133^(low)CDDP²5/5 100 CD133^(low)Saline³ 0/5 100 ¹EV, empty vector. ²CDDP, cellstreated with CDDP. ³Saline, cells treated with saline.

Transfection of miR-133a also increased messenger RNA (mRNA) levels ofthe molecules that were upregulated in CD133^(high) cells (see FIG. 6)but not CD133 mRNA, suggesting that miR-133a does not affect theexpression of the molecules on the upstream pathway of CD133 (FIG. 17).These results revealed that miR-133a is a candidate miRNA that canregulate the phenotypes of osteosarcoma TICs. Indeed, the expression ofmiR-133a was also high in the CD133^(high) fraction of osteosarcomabiopsies (FIG. 18), and high expression of miR-133a was significantlycorrelated with poor prognosis of patients (Table 10).

TABLE 10 Uni- and multivariate analyses and the relationship betweenclinicopathologic variables and miR-133a expression in 48 casesCorrelation Number of miR-133a miR-133a (CD133) χ2 Variable cases LowHigh (P value) Age (years) 0.228 0-10 9 9 0 11-20 30 23 7 21+ 9 8 1Gender 1.000 Male 31 26 5 Female 17 14 3 Site 0.566 Femur 26 22 4 Tibia16 14 2 Humerus 2 1 1 Other 4 3 1 Histology 0.142 Osteoblastic 25 23 2Chondroblastic 7 6 1 Fibroblastic 2 2 0 Other, NA* 14 9 5 Metastasis at0.330 diagnosis Present 7 5 2 Absent 41 35 6 Neoadjuvent 0.902chemotherapy MTX + DOX/CDDP 29 24 5 IFO + DOX/CDDP 18 15 3 Other 1 1 0Response to 0.173 neoadjuvent chemotherapy Good 17 16 1 (necrosis > 90%)Poor 26 21 5 (necrosis < 90%) NA* 5 3 2 miR-133a expression High 8 0 8Low 40 40 0

Example 6 Transfection of CD133^(low) SaOS2 Cells with miRNAs Results inIncreased Proliferation

CD133^(low) SaOS2 cells were isolated by cell sorting and thentransfected with negative control (NC) RNA, miR-1 alone, miR-10b alone,miR-133a alone, miR-1 plus mirR-10b, mirR-10b plus miR-133a, miR-1 plusmiR-133a, or miR-1 plus miR-10b plus miR-133a. CD133^(high) cells alsowere isolated by cell sorting and then transfected with negative control(NC) RNA. Each population of cells was separately maintained in tissueculture for 3-7 days and then studied with a light microscope to assesscell proliferation. Results are shown in FIG. 19. As is evident fromFIG. 19, CD133^(low) SaOS2 cells transfected with miR-1 alone, miR-10balone, miR-133a alone, miR-1 plus mirR-10b, mirR-10b plus miR-133a,miR-1 plus miR-133a, or miR-1 plus miR-10b plus miR-133a proliferated toa greater extent than did CD133^(low) SaOS2 cells transfected withnegative control RNA. CD133^(low) SaOS2 cells transfected with miR-1plus miR-10b plus miR-133a proliferated to nearly the same extent asCD133^(high) cells transfected with negative control RNA. The effects ofthe combinations were at least additive.

Similar results were obtained in an experiment with MNNG/HOS cells inplace of SaOS2 cells (FIG. 20).

Example 7 Transfection of CD133^(low) SaOS2 Cells with miRNAs Results inDrug Resistance

CD133^(low) SaOS2 cells were isolated by cell sorting and thentransfected with negative control (NC) RNA, miR-1 alone, miR-10b alone,miR-133a alone, or miR-1 plus miR-10b plus miR-133a, similar toExample 1. Each population of transfected cells was then separatelymaintained in tissue culture for four days in the presence of 30 nMdoxorubicin, 2.5 μM cisplatin, or 320 nM methotrexate, and then cellswere counted. Results are shown in FIG. 21. As is evident from FIG. 21,CD133^(low) SaOS2 cells transfected with miR-133a alone or with miR-1plus miR-10b plus miR-133a proliferated to a greater extent thannegative control in the presence of cisplatin and in the presence ofmethotrexate. The addition of miR-133a thus was associated withincreased resistance to cisplatin and methotrexate.

Example 8 miR-1, miR-10b, and miR-133a are Induced by CisplatinTreatment

The expressions of miR-1, miR-10b, and miR-133a, as well as CD133, wereinduced by cisplatin treatment. qRT-PCR analysis showed that DOX-treatedor CDDP-treated (3 days) 143B cells expressed an increased level ofmiR-1, miR-10b, and miR-133a relative to untreated 143B cells (FIG. 22).In addition, the expression of miR-133a was enhanced by cisplatin inCD133^(low) HOS cells. Furthermore, exposure to CDDP increased in vivotumorigenicity of CD133^(low) HOS population. CDDP-treated CD133^(low)HOS cells could form tumors with as few as 100 cells per injection,whereas the untreated CD133^(low) HOS cells could not (FIG. 23 and Table9). These data indicate that the TIC phenotypes, as well as theexpression of CD133 mRNA and miR-133a, might be enhanced bychemotherapeutics. Therefore, we reasoned that silencing of miR-133abefore or during chemotherapy would prevent the increase of theexpression of miR-133a, which enhanced TIC phenotypes and was induced bychemotherapeutics.

Example 9 Antisense to miR-133a Reduces Proliferation of CD133^(high)Cells

To evaluate whether silencing of miR-133a can suppress malignantphenotypes of osteosarcoma, experiments opposite those of Example 8 wereperformed by introducing locked nucleic acid (LNA) anti-miR-133a. LNA isa class of nucleic acid analogs possessing very high affinity andexcellent specificity toward complementary DNA and RNA, and LNAoligonucleotides have been applied as antisense molecules both in vitroand in vivo. Elmèn, J et al. (2008) Nature 452:896-9; Obad, S et al.(2011) Nat. Genet. 43:371-8. CD133^(high) population of SaOS2 and HOScells was isolated by cell sorting and transfected withLNA-anti-miR-133a (LNA-133a) and LNA-negative control (LNA-NC). As acontrol, the isolated CD133^(low) SaOS2 and HOS cells were alsotransfected with LNA-NC. The efficacy of LNA-133a for the silencing ofmiR-133a was confirmed by real-time RT-PCR analysis (FIG. 24).CD133^(high) SaOS2 and HOS cells transfected with LNA-133a weresuppressed in proliferation rate to the same level of CD133^(low) cellstransfected with LNA-NC (FIG. 25).

Example 10 LNA-133a-Transduced CD133^(high) Cells Exhibit Enhanced DrugSensitivity and Decreased Invasiveness

LNA-133a-transduced CD133^(high) cells exhibited enhanced sensitivity toDOX and CDDP at the level of LNA-NC-transduced CD133^(low) cells. Theseresults were validated by counting Hoechst-stained cells showingapoptotic nuclear condensation and fragmentation in CD133^(high) cells.There was a significantly higher apoptotic cell death rate inLNA-133a-transduced CD133^(high) cells compared to control CD133^(high)cells (FIG. 26). Furthermore, LNA-133a decreased the invasiveness ofCD133^(high) SaOS2 and HOS populations (FIG. 27) and the expression ofthe molecules associated with CD133^(high) phenotypes (FIG. 28).Collectively, these observations suggest that silencing of miR-133a inCD133^(high) cells can reduce the malignant phenotype of osteosarcomaTICs, including drug resistance and invasion.

Example 11 Silencing of miR-133a In Vivo is Effective for the Treatmentof Osteosarcoma and Exhibits Synergistic Efficacy in Combination withCDDP

To extend the in vitro findings and to determine whether silencing ofmiR-133a could be an effective therapeutic option for osteosarcomatreatment, the effect of LNA-133a on a spontaneous lung metastasis modelof osteosarcoma was examined. Experimentally, 1.5×10⁶ cells of 143Btransfected with firefly luciferase gene (143B-luc) were implantedorthotopically into the right proximal tibia of athymic nude mice. Theimplanted tumor growth and the presence of distant metastases wereanalyzed weekly for luciferase bioluminescence using an in vivo imagingsystem (IVIS). A new treatment protocol was made of LNA-133a intravenous(i.v.) administration (10 mg kg⁻¹) 24 h before intraperitoneal (i.p.)injection of CDDP (2.5 mg kg⁻¹) (FIG. 29) in order to decrease drugresistance and to prevent the induction of TIC phenotypes bychemotherapy, which were observed in in vitro experiments. Before theanimal study of this protocol, we confirmed reduced miR-133a levels inosteosarcoma tissues from LNA-133a-treated mice compared with those fromsaline-treated mice (FIG. 30). To assess the efficacy of the newtreatment protocol, results were compared with three control groups(n=10 each): a saline control group, an LNA group, and a CDDP group. At5 weeks, half the mice in each group were euthanized for furtheranalysis; the remaining mice were monitored for survival.

Results. The expression of miR-133a of tumors was decreased in thepresence of LNA-133a (FIG. 31). Mice that had been administered LNA-133a(10 mg kg⁻¹ i.v.) and CDDP (2 mg kg⁻¹ i.p.) showed a significantlysmaller tumor growth compared to the other groups (FIGS. 32 and 33). Nosignificant differences in tumor growth were observed in the presence orin the absence of CDDP alone (FIGS. 32 and 33). Furthermore, lungmetastasis was observed in 10/10 (100%) of the saline group, 7/10 (70%)of the LNA group, 8/10 (80%) of CDDP group, and only 3/10 (30%) ofcombination group (LNA-133a+CDDP) on day 35 (Table 11).

TABLE 11 Outcome of LNA treatment in osteosarcoma-bearing mice TumorWeight Lung Luminescence Group (mean) (g) metastasis of lung (mean)ctrl. 3.928 10/10 5047 LNA 3.143  7/10 1744 CDDP 3.957  8/10 2855 LNA +CDDP 1.901  3/10 582

The average luminescence at chest region was significantly decreased inmice treated with the combination of LNA-133a and CDDP. Both the numberand size of lung metastasis at every lobe were validated in theluciferase assay and histopathological examination.

Notably, the effect of the combination therapy (LNA-133a+CDDP) was foundto exhibit synergistic inhibition of lung metastasis.

Furthermore, the combination therapy (LNA-133a+CDDP) significantlyextended the survival period of tumor-bearing mice (log-rank test,P=0.0084, FIG. 34). Despite the highly conserved sequence of maturehuman miR-133a and murine miR-133a (e.g., GenBank Accession No.NR_029676; 5′-UUUGGUCCCCUUCAACCAGCUG-3′; SEQ ID NO:3), all mice showedminimal toxic effects on various tissue including heart, liver, andskeletal muscle during the observation period. Thus, systemicadministration of LNA-133a is effective for suppression of tumor growthand lung metastasis in the xenograft model for highly metastaticosteosarcoma in the presence of cisplatin.

Example 12 Identification of Gene Targets for miR-133a

The examples above establish that miR-133a regulates the malignancy ofCD133^(high) osteosarcoma TICs, and inhibition of miR-133a expression inosteosarcoma cells inhibits the tumor development. In order tounderstand the mechanisms regulated by miR-133a in CD133^(high)osteosarcoma TICs, candidate mRNA expression profiling was performed bytwo different microarray analyses together with in silico predictions(FIG. 35). We detected 1812 downregulated genes with at least a 1.2-folddecrease in the first microarray analysis of total RNA collected fromSaOS2 CD133^(low) cells transduced miR-133a or NC, whereas 4976upregulated genes were detected with at least a 2.0-fold increase in thesecond microarray analysis of mRNA expression in RNA collected fromanti-Ago2 antibody immunoprecipitation in CD133^(low) cells transducedwith miR-133a or NC (FIG. 36). Subsequently, 226 genes were collected byboth methods (Table 12), and 20 genes were identified in TargetScanHuman6.0, one of the publicly available in silico databases (FIG. 36).

TABLE 12 Predicted gene targets for miR-133a by two analyses of cDNAmicroarray and in silico prediction Fold increase, Fold decrease,GenBank miR-133a- miR-133a Gene Symbol Accession Gene Name Ago2 complextransfection PGAP1 NM_24989 post-GP1 attachment to proteins 1 7.60 −0.42C1orf118 XR_041258 chromosome 1 open reading frame 118 7.19 −0.28 DYNLT3NM_006520 dynein, light chain, Tetex-type 3 7.17 −0.42 AGFG1NM_001135187 ArtGAP with FG repeats 1 6.69 −0.28 WDR44 NM_019045 WDrepeat domain 44 6.36 −0.43 FLYWCH1 NM_020912 FLYWCH-type zinc finger 15.62 −0.32 CARKD NM_018210 carbohydrate kinase domain containing 5.50−0.66 CUL4B NM_003588 cullin 4B 5.30 −0.40 ETS1 NM_005238 v-etserythroblastosis virus E26 5.29 −0.74 HDAC6 NM_006044 histonedeacetylase 6 5.20 −0.36 C19orf10 NM_019107 chromosome 19 open readingframe 10 5.17 −0.36 RASA2 NM_006506 RAS p21 protein activator 2 5.15−0.44 KIAA1958 NM_133465 KIAA1958 5.14 −0.28 LOC645851 NR_024395hypothetical LOC645851 5.09 −0.37 TBPL1 NM_004865 TBP-like 1 4.86 −0.33SNX26 NM_052948 sorting nexin 26 4.73 −0.27 SCRN1 NM_001145513 secemin 14.72 −0.55 LOC100132672 XR_038504 similar to glycosyltransferase 8domain 4.61 −0.36 containing 3 AP4S1 NM_001128126 adaptor-relatedprotein complex 4, 4.39 −0.59 sigma 1 subunit SF3B3 NM_012426 splicingfactor 3b, subunit 3, 130 kDa 4.34 −0.27 LMBR1 NM_022458 limb region 1homolog (mouse) 4.31 −0.40 HERC4 NM_022079 hect domain and RLD 4 4.26−0.69 ADAMTS1 NM_006988 ADAM metallopeptidase with 4.17 −0.47thrombospondin type 1 motif, 1 CYB5R4 NM_016230 cytochrome b5 reductase4 4.14 −0.29 LHFPL2 NM_005779 lipoma HMGIC fusion partner-like 2 4.11−0.30 FAM13AOS NR_002806 FAM13A opposite strand (non-protein 4.04 −0.28coding) RALGAPA1 NM_1940301 Ral GTPase activating protein, alpha 4.00−0.53 subunit 1 (catalytic) TNFRSF13C NM_052945 tumor necrosis factorreceptor 3.99 −0.44 superfamily, member 13C LOC100131829 AK124002hypothetical protein LOC100131829 3.95 −0.43 C1orf58 NM_144695chromosome 1 open reading frame 58 3.92 −0.54 TNFRSF10D NM_003840 tumornecrosis factor receptor 3.75 −0.45 superfamily, member 10d, decoy withtruncated death domain ZDHHC17 NM_015336 zinc finger, DHHC-typecontaining 17 3.68 −0.36 LOC729603 NR_003288 calcium binding protein P223.62 −0.34 pseudogene SNX30 NM_001012994 sorting nexin family member 303.55 −0.41 TSTD2 NM_139246 thiosulfate sulfurtransferase 3.52 −0.38(rhodanese)-like domain containing 2 SPRYD4 NM_207344 SPRY domaincontaining 4 3.51 −0.35 PTPMT1 NM_175732 protein tyrosine phosphatase,3.50 −0.38 mitochondrial 1 KLHDC4 NM_017566 kelch domain containing 43.43 −0.43 SLC30A7 NM_133496 solute carrier family 30 (zinc 3.38 −0.48transporter), member 7 TCEA3 NM_003196 transcription clongation factor A(SII), 3 3.32 −0.39 GORASP1 NM_031899 golgi reassembly stacking protein1, 3.17 −0.51 65 kDa RBM15B NM_013286 RNA binding motif protein 15B 3.13−0.80 PDGFRB NM_002609 platelet-derived growth factor receptor, 3.13−0.37 beta polypeptide ITPRIPL2 NM_001034841 inositol 1,4,5-triphosphatereceptor 3.09 −0.34 interacting protein-like 2 FBXO3 NM_033406 F-boxprotein 3 3.01 −0.35 FAM122B NM_001166600 family with sequencesimilarity 122B 3.00 −0.30 MINPP1 NM_004897 multiple inositolpolyphosphate 2.98 −0.28 histidine phosphatase, 1 SPOPLB NM_001001664speckle-type POZ protein-like 2.94 −0.47 FAM86B1 NM_001083537 familywith sequence similarity 86, 2.85 −0.44 member B1 LOC100138071XM_001724939 similar to hCG41624 2.83 −0.31 CCNT1 NM_001240 cyclin T12.82 −0.53 AP2M1 NM_004068 adaptor-related protein complex 2, mu 1 2.80−0.44 subunit AKIRIN1 NM_024595 akirin 1 2.73 −0.27 CHMP5 NM_016410chromatin modifying protein 5 2.69 −0.61 PPM1K NM_152542 proteinphosphatase 1K (PP2C domain 2.67 −0.53 containing) MICALL2 NM_182924MICAL-like 2 2.66 −0.29 DGKZ AK123378 diacylglycerol kinase, zeta 104kDa 2.65 −0.39 SCARNA16 NR_003013 small Cajal body-specific RNA 16 2.65−0.50 SERPINE1 NM_000602 serpin peptidase inhibitor, clade E 2.65 −0.61(nexin, plasminogen activator inhibitor type 1), member 1 STARD13NM_178006 StAR-related lipid transfer (START) 2.58 −0.28 domaincontaining 13 ROD1 NM_005156 ROD1 regulator of differentiation 1(S. 2.55−0.36 pombe) VEGFC NM_005429 vascular endothelial growth factor C 2.45−0.35 MYH9 NM_002473 myosin, heavy chain 9, non-muscle 2.43 −0.39 CCNJNM_019084 cyclin J 2.41 −0.26 WDR66 NM_144668 WD repeat domain 66 2.38−0.32 CSRNP1 NM_033027 cysteine-serine-rich nuclear protein 1 2.34 −0.28MYBL1 NM_001144755 v-myb myeloblastosis viral oncogene 2.32 −0.29homolog (avian)-like 1 EME2 BC041011 essential meiotic endonuclease 12.31 −0.44 homolog 2 (S. pombe) MGC27382 AK091757 hypothetical MGC273822.27 −0.33 MMP14 NM_004995 matrix metallopeptidase 14 (membrane- 2.27−0.42 inserted) WASH1 NM_182905 WAS protein family homolog 1 2.26 −0.32RIMS2 NM_014677 regulating synaptic membrane 2.25 −0.45 exocytosis 2HSPG2 NM_005529 heparan sulfate proteoglycan 2 2.25 −0.45 HDAC8NM_018486 histone deacetylase 8 2.21 −0.34 AK2 NM_013411 adenylatekinase 2 2.20 −0.30 SRRM2 NM_016333 serine/arginine repetitive matrix 22.19 −0.28 LOC731419 XM_001132610 hypothetical protein LOC731419 2.19−0.32 SYNGR3 NM_004209 synaptogyrin 3 2.11 −0.33 PRUNE2 NM_015225 prunehomolog 2 (Drosophila) 2.11 −0.38 MLKL NM_152649 mixed lineage kinasedomain-like 2.10 −0.30 DST NM_001723 dystonin 2.10 −0.44 PBXIP1NM_020524 pre-B-cell leukemia homeobox 2.03 −0.78 interacting protein 1ANTXR2 NM_058172 anthrax toxin receptor 2 1.98 −0.61 NSF NM_006178N-ethylmaleimide-sensitive factor 1.98 −0.32 AP1HA NM_001077628 anteriorpharynx dtective 1 homolog A 1.98 −0.34 (C. elegans) RASA1 NM_002890 RASp21 protein activator (GTPase 1.95 −0.46 activating protein) 1 BAIAP2NM_017451 BAII-associated protein 2 1.91 −0.29 GARNL3 NM_032293 GTPaseactivating Rap/RanGAP 1.90 −0.49 domain-like 3 CKLF NM_016951chemokine-like factor 1.89 −0.38 SNORD17 NR_003045 small nucleolar RNA,C/D box 17 1.88 −0.27 TRIT1 NM_017646 tRNA isopentenyltransferase 1 1.88−0.43 FILIP1L NM_182909 filamin A interacting protein 1-like 1.88 −0.36VAMP2 NM_014232 vesicle-associated membrane protein 2 1.87 −0.31(synaptobrevin 2) TBL1X NM_005647 transducin (beta)-like 1X-linked 1.87−0.30 LOC729314 XR_037423 similar to POM121-like protein 1 1.85 −0.63RHD NM_016124 Rh blood group, D antigen 1.84 −0.28 HERC2 NM_004667 hectdomain and RLD 2 1.81 −0.36 KIAA1967 NM_021174 KIAA1967 1.78 −0.32 YIPF2NM_024029 Yip1 domain family, member 2 1.71 −0.48 MLL5 NM_182931myeloid/lymphoid or mixed-lineage 1.71 −0.41 leukemia 5 (trithoraxhomolog, Drosophila) DUSP11 NM_003584 dual specificity phosphotase 111.70 −0.28 (RNA/RNP complex 1-interacting) ABL2 NM_001100108 v-ablAbelson murine leukemia viral 1.68 −0.30 oncogene homolog 2 (arg,Abelson- related gene) RBMX2 NM_016024 RNA binding motif protein,X-linked 2 1.65 −0.37 ALS2CR8 NM_024744 amyotrophic lateral selerosis 21.65 −0.30 (juvenile) chromosome region, candidate 8 IDH1 NM_005896isocitrate dehydrogenase 1 (NADP+), 1.65 −0.31 soluble NT5C3L NM_0529355′-nucleotidase, cytosolic III-like 1.63 −0.40 ERMP1 NM_024896endoplasmic reticulum metallopeptidase 1 1.59 −0.86 GPSM1 NM_015597G-protein signaling modulator 1 1.53 −0.47 (AGS3-like, C. elegans)ARHGEF10L NM_018125 Rho guanine nucleotide exchange factor 1.52 −0.32(GEF) 10-like MFN2 NM_014874 mitofusin 2 1.52 −0.32 CG030 NR_026928hypothetical CG030 1.49 −0.30 UBXN7 NM_015562 UBX domain protein 7 1.49−0.35 CCDC45 NM_138363 coiled-coil domain containg 45 1.47 −0.39 ZNF701NM_018260 zinc finger protein 701 1.46 −0.68 LOC642406 AK024257 similarto contactin associated protein- 1.46 −1.34 like 3B PHF8 NM_015107 PHDfinger protein 8 1.44 −0.28 MED23 NM_015979 mediator complex subunit 231.42 −0.29 ARHGAP11B NM_001039841 Rho GTPase activatingprotein 11B 1.42−0.29 MYST4 NM_012330 MYST histone acetyltransferase 1.41 −0.31(monocytic leukemia) 4 SYT17 NM_016524 synaptotagmin XVII 1.40 −0.39DPM2 NM_003863 dolichyl-phosphate mannosyltransferase 1.30 −0.35polypeptide 2, regulatory subunit TUB NM_003320 tubby homolog (mouse)1.30 −0.27 TBPL1 NM_004865 TBP-like 1 1.30 −0.28 FAM40B NM_020704 familywith sequence similarity 40, 1.30 −0.62 member B DOLPP1 NM_020438dolichyl pyrophosphate phosphatase 1 1.29 −0.34 HIST1112BM NM_003521histone cluster 1, 112bm 1.29 −0.35 ZBTB7A NM_015898 zinc finger and BTBdomain containing 7A 1.28 −0.28 SLC30A7 NM_133496 solute carrier family30 (zinc 1.25 −0.36 transporter), member 7 HCRTR1 NM_001525 hypocretin(orexin) receptor 1 1.22 −0.27 DNAJB6 NM_005494 DnaJ (Hsp40) homolog,subfamily B, 1.22 −0.56 member 6 QPRT NM_014298 quinolinatephosphoribosyltranferase 1.20 −0.32 CCDC75 NM_174931 coiled-coil domaincontaining 75 1.20 −0.29 NPTXR NM_014293 neuronal pentraxin receptor1.19 −0.63 RHOB NM_004040 ras homolog gene family, member B 1.19 −0.48CDH7 NM_004361 cadherin 7, type 2 1.17 −0.34 COL5A1 AK057231 collagen,type V, alpa 1 1.17 −0.50 SGMS2 NM_152621 sphingomyelin synthase 2 1.16−0.32 LOC643802 XM_001716860 similar to M-phase phosphoprotein 10 1.15−0.53 (U3 small nucleolar ribonucleoprotein) BCL11A NM_018014 B-cellCLL/lymphoma 11A (zinc finger 1.14 −0.28 protein) GEN1 NM_182625 Genhomolog 1, endonuclease 1.14 −0.28 (Drosphila) ZP1 NM_207341 zonapellucida glycoprotein 1 (sperm 1.12 −0.30 receptor) EFTUD1 NM_024580elongation factor Tu GTP binding 1.12 −0.47 domain containing 1 REEP6NM_138393 receptor accessory protein 6 1.10 −0.58 UBA2 NM_005499ubiquitin-like modifier activating 1.08 −0.38 enzyme 2 BRIP1 NM_032043BRAC1 interacting protein C-terminal 1.06 −0.27 helicase 1 KPTNNM_007059 kaptin (actin binding protein) 1.06 −0.57 DZIP1 NM_014934 DAZinteracting protein 1 1.04 −0.35 MGC16275 NR_026914 hypothetical proteinMGC16275 1.04 −0.29 APTX NM_017692 aprataxin 1.03 −0.29 P2RX4 NM_002560purinergic receptor P2X, ligand-gated 1.02 −0.29 ion channel, 4 PCDH24NM_017675 protocadherin 24 1.00 −0.37

Overall, ten putative candidates for miR-133a target genes were selectedwith these data combined. Next, the expression of these molecules wasreduced using siRNA-induced gene knockdown system to investigate whetherthese candidates are functionally important targets of miR-133a inosteosarcoma cells. As a result, knockdown of four candidates (ANXA2,DUSP11, MAST4, and ROD1) in CD133^(low) SaOS2 cells enhanced drugresistance (FIG. 37), and knockdown of five candidates (ANXA2, DUSP11,SGMS2, SNX30, and UBA2) enhanced invasiveness of CD133^(low) SaOS2 cells(FIG. 38).

Of course, the effect of knockdown of these putative target genes wouldbe similar to the effect exerted by miR-133a on these same genes,resulting in enhanced drug resistance and enhanced invasiveness ofCD133^(low) SaOS2 cells. Conversely, silencing of miR-133a inCD133^(low) SaOS2 cells would be expected to be permissive forexpression of the putative target genes, thereby reducing drugresistance and reducing invasiveness of CD133^(low) SaOS2 cells.

To validate whether these molecules are regulated by miR-133a, the 3′UTR(untranslated region) fragment containing putative miR-133a bindingsites was cloned downstream of a luciferase coding sequence, and theluciferase reporter and miR-133a oligonucleotides were co-transfectedinto SaOS2 cells. As a control, the luciferase reporter and NColigonucleotides were co-transfected into SaOS2 cells. Luciferaseactivities were reduced by approximately 39-73% in the cellsco-transfected with miR-133a compared with the cells co-transfected withthe NC oligonucleotides (FIG. 39). From results of this assay, ANXA2,DUSP11, MAST4, SGMS2, SNX30, and UBA2 were found to function as directtargets of miR-133a.

Indeed, these target genes or their family genes were previouslysuggested to function as tumor suppressors in certain other cancers.Gostissa, M et al. (1999) EMBO J. 18:6462-71; Caprara, G et al. (2009)J. Cell. Mol. Med. 13:2158-70; Nguyen, L N et al. (2006) Clin, CancerRes. 12:6952-9. ANXA2 has been reported to be associated withtumor-suppressive function in osteosarcoma (Gillette, J M (2004) J. CellBiochem. 92:820-32), whereas MAST4 has been unknown in tumor biology(Garland, P (2008) Brain Res. 21:12-19).

Indeed, as disclosed by the present invention, the expression of allthese targets was increased by silencing of miR-133a in CD133^(high)cells (FIG. 40) and decreased by miR-133a upregulation in CD133^(low)cells (FIG. 41), consistent with observed inverse correlations with theexpression of CD133 and miR-133a in both xenografted tumors (FIG. 42)and clinical samples (results not shown). The increased expression ofthe targets by silencing of miR-133a was confirmed by qRT-PCR (resultsnot shown) and immunohistochemistry of LNA-treated tumors (results notshown). Finally, in investigating the relationships between theexpression of these targets and osteosarcoma patient prognosis, astrikingly close correlation was found between the mRNA expression ofthe miR-133a targets and patient prognosis (FIG. 43). Patients withhigher expression levels of these targets survived much longer thanpatients with lower expression, indicating that these targets couldfunction as novel tumor-suppressors in osteosarcoma.

1-21. (canceled)
 22. A method of screening for osteosarcoma, comprising: performing on a tissue sample from a subject an assay specifically capable of detecting at least one microRNA (miRNA) selected from the group consisting of miR-1, miR-10b, and miR-133a, wherein detection by the assay of the presence in the sample of the at least one miRNA indicates the subject is at risk of having osteosarcoma.
 23. The method of claim 22, wherein the tissue is blood.
 24. The method of claim 22, wherein the tissue is serum. 25-27. (canceled)
 28. The method of claim 22, wherein the miRNA is miR-1.
 29. The method of claim 22, wherein the miRNA is miR-10b.
 30. The method of claim 22, wherein the miRNA is miR-133a.
 31. The method of claim 22, wherein the assay comprises RT-PCR using oligonucleotide primers suitable for reverse transcribing and amplifying at least one miRNA selected from the group consisting of miR-1, miR-10b, and miR-133a.
 32. The method of claim 31, wherein the miRNA is miR-1.
 33. The method of claim 31, wherein the miRNA is miR-10b.
 34. The method of claim 31, wherein the miRNA is miR-133a.
 35. The method of claim 22, further comprising the step of verifying the presence of osteosarcoma in the subject when the subject is determined to be at risk of having osteosarcoma.
 36. The method of claim 35, wherein verifying the presence of osteosarcoma in the subject comprises performing on the subject a diagnostic procedure selected from the group consisting of skeletal survey or specific bone imaging analysis using X-rays or other suitable bone imaging technique, magnetic resonance imaging (MRI), computed tomography (CT), biopsy, and any combination thereof.
 37. The method of claim 35, wherein the miRNA is miR-1.
 38. The method of claim 35, wherein the miRNA is miR-10b.
 39. The method of claim 35, wherein the miRNA is miR-133a.
 40. The method of claim 35, further comprising the step of treating the subject for osteosarcoma.
 41. The method of claim 22, further comprising the step of treating the subject for osteosarcoma. 